Recombinant human naglu protein and uses thereof

ABSTRACT

The present invention provides compositions comprising an isolated mixture of recombinant human NaGlu proteins in which a substantial amount of the NaGlu proteins in the mixture has increased levels of phosphorylated mannose that confer the proteins to be efficiently internalized into human cells. The present invention also provides methods of producing such mixture of NaGlu proteins, vectors used in transgenesis and expression, host cells harboring such vectors, and methods of isolating and purifying the mixture of NaGlu proteins. The invention further provides methods of treating NaGlu associated diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related and claims priority to U.S. ProvisionalApplication Ser. No. 61/546,248, filed Oct. 12, 2011, the entirecontents of which are expressly incorporated herein by this reference.

BACKGROUND OF THE INVENTION

Sanfilippo Syndrome B is an autosomal recessive lysosomal storagedisease (LSD) caused by a deficiency in a lysosomal enzyme known asN-acetyl-alpha-D-glucosaminidase (NaGlu). NaGlu is required for thedegradation of heparan sulfate as part of the stepwise breakdown ofglycosaminoglycans (GAG) in the lysosome. The deficiency or absence ofNaGlu leads to accumulation and urinary excretion of heparan sulfate.With over 70 different mutations identified to date, Sanfilippo SyndromeB exhibits extensive molecular and genetic heterogeneity.

Approximately 1 out of 200,000 births is affected by Sanfilippo SyndromeB and the deficiency mainly manifests in young children. After initialsymptom-free interval, patients suffering from Sanfilippo Syndrome Busually present with a slowing of mental development and behavioralproblems, followed by progressive intellectual decline resulting insevere mental retardation, dementia and motor disease. Acquisition ofspeech is slow and incomplete. Profoundly affected patients may presentdelayed psychomotor and speech development as early as 2 years of age.The disease usually progresses to increasing behavioral disturbance andsleep disturbance. Although the clinical features are mainlyneurological, patients often develop diarrhea, carious teeth, anenlarged liver and spleen, stiff joints, hirsteness and/or coarse hairand may exhibit blood-clotting problems. In the final stage of theillness, patients become immobile and unresponsive and developswallowing difficulties and seizure. The life-span of an affected childtypically does not extend beyond late teens to early twenties.

Different approaches have been attempted to provide the missing enzymein patients. To produce NaGlu for enzyme replacement therapy (ERT),human NaGlu has been expressed in various mammalian cell culturesystems. However, in contrast to the naturally occurring NaGlu whichtrafficks to the lysosome intracellularly, recombinant NaGlu proteinsproduced and secreted from mammalian cells were found to contain no oronly a trace amount of mannose 6-phosphate (M6P). The absence orscarcity of M6P moieties in the secreted NaGlu has been known to preventits efficient internalization into target cells (e.g., human skinfibroblasts), which have M6P receptors on the surface on its plasmamembrane (see, Zhao et al., Protein Expression and Purification,19:202-211 (2000); and Weber et al., Protein Expression andPurification, 21:251-259 (2001)). The low degree of phosphorylation wasseen in secreted mouse NaGlu expressed in CHO cells, secreted humanNaGlu expressed in HeLa cells, secreted human NaGlu expressed in humanfibroblasts, and secreted human NaGlu expressed in human embryonickidney (HEK) cell line 293 (see, Zhao et al., Protein Expression andPurification, 19:202-211 (2000); Yogalingam et al., Biochim Biophys.Acta 1502: 415-425; and Weber et al., Protein Expression andPurification, 21:251-259 (2001)). No or weak phosphorylation ofN-glycans in the NaGlu proteins secreted from the mammalian cells hasposed a major obstacle for the development of a recombinant human NaGluprotein suitable for enzyme replacement therapy as all theaforementioned attempts has failed to produce an enzyme which isefficiently taken up by target cells as the concentration of theinternalized proteins, if detectable at all, was nearly a thousand timesless than wild-type levels (see, Zhao et al., Protein Expression andPurification, 19:202-211 (2000)). To date, no approved product isavailable for the treatment of Sanfilippo Syndrome B.

Direct administration of mammalian cell-produced recombinant human NaGluprotein (rhNaGlu) having the native amino acid sequence into the centralnervous system (CNS) (e.g., intrathecal administration into thecerebrospinal fluid (CSF)) of NaGlu deficient mice has been attempted,but failed to demonstrate successful biodistribution of the enzyme tothe brain due to excessive accumulation of the protein on the ependymalling of the ventricles as well as lack of requisite M6P residues forefficient cellular uptake. Similarly, systemic administration (i.e.,intravenous (IV) injection) of mammalian cell-produced rhNaGlu havingthe native amino acid sequence also failed to demonstrate successfullocalization of the protein to the brain. In addition to known risksassociated with highly invasive intrathecal administration, theseobstacles in targeting rhNaGlu to the brain have been too great achallenge to achieve effective therapy for the treatment of SanfilippoSyndrome B.

Therefore, there is a need to provide a stable NaGlu protein which isenzymatically active and has physical properties that allow for theprotein to cross the blood brain barrier (BBB) and for effectiveinternalization of the protein into the lysosomes of target cells. Thereis also a need for a high expressing and robust protein productionplatform which can provide a recombinant human NaGlu that effectivelycrosses the blood brain barrier and is efficiently internalized intohuman target cells.

SUMMARY OF THE INVENTION

The present invention is drawn to compositions comprising recombinanthuman NaGlu protein (rhNaGlu) useful for therapy, for example, in thetreatment of Sanfilippo Syndrome B. The present invention is based onthe surprising and unexpected discovery that the rhNaGlu describedherein has one or more glycosylation patterns that allow the rhNaGlu toefficiently cross the blood brain barrier (BBB), and be taken up intocells within the central nervous system (CNS) of animals deficient inthe enzyme, resulting in a dramatic increase inα-N-acetylglucosaminidase activity in the brain, as well as a reductionof substrate levels. Moreover, the rhNaGlu described herein isefficiently taken up into a mammalian cell (e.g., human cell), resultingin an increased enzymatic activity as compared to NaGlu proteinsproduced and secreted from unmodified mammalian cells that are notdesigned to produce specific glycosylation. The increased cellularuptake of the NaGlu protein also provides benefits for the use in enzymereplacement therapy for a human patient suffering from SanfilippoSyndrome B by minimizing the need for an increased amount and frequencyof dose, and thereby greatly reducing the potential risk ofimmunogenicity.

The rhNaGlu protein described herein contains sufficient amount ofoligosaccharides (e.g., mannose and phosphorylated mannose (i.e., M6P))to allow efficient cellular uptake via mannose and/or M6Preceptor-mediated endocytosis and be correctly targeted into humancells. In one embodiment, the rhNaGlu contains at least one mole ofprotein, for example, 1, 2, 3, 4, 5 or 6 moles of M6P per mole ofprotein. In one embodiment, rhNaGlu can be internalized into a NaGludeficient human cell such that the internalized protein fully (100% ormore) restores normal levels (i.e., wild-type levels) of NaGlu activityin the NaGlu deficient cell.

Also disclosed herein are methods for producing a transgenic avian thatexpresses rhNaGlu which benefits from phosphorylation of mannose. Inparticular, a transgenic avian that expresses rhNaGlu protein in oviductcells, secretes into the lumen of the oviduct and deposits the proteininto egg white. Avian eggs that contain such rhNaGlu are also includedin the present invention.

The present invention also contemplates vectors and host cells thatcontain a transgene encoding rhNaGlu as well as pharmaceuticalcompositions comprising rhNaGlu to be used in the application of suchrhNaGlu for the treatment of Sanfilippo Syndrome B.

In one aspect, the invention provides a composition comprising anisolated mixture of recombinant human N-acetyl-alpha-D-glucosaminidase(rhNaGlu) comprising the amino acid sequence 24-743 of SEQ ID NO:1,wherein at least 10% of the rhNaGlu in the mixture comprises at leastone glycan structure having mannose-6-phosphate (M6P). In oneembodiment, the rhNaGlu having M6P is capable of being taken up into amammalian cell deficient in NaGlu such that internalized rhNaGlurestores at least 50%, 60%, 70%, 80%, 90% or 100% of normal NaGluactivity observed in a wild-type mammalian cell of the same type. Inanother embodiment, the glycan structure is an N-linked glycan.

In one embodiment, the rhNaGlu contains at least 1 mole of M6P per moleof protein. In another embodiment, the rhNaGlu contains between about 1and about 6 moles of M6P per mole of protein. In another embodiment, therhNaGlu contains about 2 moles of M6P per mole of protein. In yetanother embodiment, the rhNaGlu contains about 3 moles of M6P per moleof protein. In another embodiment, the rhNaGlu contains about 4 moles ofM6P per mole of protein. In another embodiment, the rhNaGlu containsabout 5 moles of M6P per mole of protein. In yet another embodiment, therhNaGlu contains about 6 moles of M6P per mole of protein.

In one embodiment, the mammalian cell deficient in NaGlu is a humancell. In another embodiment, the human cell deficient in NaGlu is a skinfibroblast, a hepatocyte or a macrophage. In one embodiment, the humancell deficient in NaGlu is a neuronal cell.

In one embodiment, the rhNaGlu is effectively delivered to the brain ofa mammal having NaGlu deficiency when systemically administered. In oneparticular embodiment, the rhNaGlu is effectively delivered to the brainof a mammal having NaGlu deficiency when intravenously administered. Inone embodiment, the rhNaGlu is effectively delivered to the brain of amammal having NaGlu deficiency when administered intrathecally.

In one embodiment, the rhNaGlu having M6P is internalized by a NaGludeficient cell and restores at least 100% of normal NaGlu activity invivo. In one embodiment, the rhNaGlu having M6P contains at least 25moles of mannose per mole of protein.

In one embodiment, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or95% of the rhNaGlu in the mixture contains M6P. In another embodiment,at least 20% of the rhNaGlu in the mixture contains at least one M6P. Inanother embodiment, at least 30% of the rhNaGlu in the mixture containsat least one M6P. In another embodiment, at least 40% of the rhNaGlu inthe mixture contains at least one M6P. In another embodiment, at least50% of the rhNaGlu in the mixture contains at least one M6P. In anotherembodiment, at least 60% of the rhNaGlu in the mixture contains at leastone M6P.

In another aspect, the invention provides a composition comprising anisolated mixture of recombinant human N-acetyl-alpha-D-glucosaminidase(rhNaGlu) comprising the amino acid sequence 24-743 of SEQ ID NO:1,wherein the mixture comprises a sufficient amount of rhNaGlu containingone or more glycan structures comprising mannose-6-phosphate (M6P) suchthat the rhNaGlu containing M6P is internalized into a mammalian cellhaving NaGlu deficiency via M6P receptor-mediated endocytosis andrestores at least 50% of NaGlu activity observed in a wild-type cell ofthe same type expressing endogenous NaGlu. In one embodiment, therhNaGlu is N-linked glycosylated. In another embodiment, the rhNaGlu isO-linked glycosylated.

In one embodiment, the rhNaGlu comprises at least 1 moles of M6P permole of rhNaGlu. In another embodiment, the rhNaGlu comprises about 1,2, 3, 4, 5 or 6 moles of M6P per mole of rhNaGlu. In another embodiment,the rhNaGlu comprises about 3 moles of M6P per mole of rhNaGlu. Inanother embodiment, the rhNaGlu comprises about 4 moles of M6P per moleof rhNaGlu.

In one embodiment, the rhNaGlu comprises mannose. In another embodiment,the rhNaGlu comprises N-acetylglucosamine (G1cNAc). In anotherembodiment, the rhNaGlu comprises galactose. In another embodiment, therhNaGlu comprises N-acetylgalactosamine (GalNAc). In another embodiment,the rhNaGlu contains no fucose. In another embodiment, the rhNaGlucontains no glucose. In one embodiment, the rhNaGlu restores at least60, 70, 80, 90, 95 or 100% of normal NaGlu enzymatic activity.

In another embodiment, the rhNaGlu is effectively delivered to the brainof a mammal having NaGlu deficiency when administered systemically. Inone embodiment, the rhNaGlu is effectively delivered to the brain of amammal having NaGlu deficiency when administered intravenously. Inanother embodiment, the rhNaGlu is effectively delivered to the brain ofa mammal having NaGlu deficiency when administered intrathecally.

In one embodiment, the mammalian cell deficient in NaGlu is a humancell. In another embodiment, the human cell is a skin fibroblast, ahepatocyte or a macrophage. In one embodiment, the human cell deficientin NaGlu is a neuronal cell.

In one embodiment, the rhNaGlu is a fusion protein comprising a secondmoiety. In one embodiment, the second moiety is a polypeptide. Inanother embodiment, the polypeptide is selected from the groupconsisting of transferrin receptor ligand (TfRL), insulin-like growthfactor receptor (IGF2R) ligand, low density lipoprotein (LDL) receptorligand and acidic amino acid (AAA) residues.

In one embodiment, the rhNaGlu is produced from a transgenic avian. Inone embodiment, the transgenic avian is a chicken, a turkey, a duck or aquail. In one embodiment, the transgenic avian is a chicken. In oneembodiment, the rhNaGlu is produced from an oviduct cell.

In another aspect, the invention provides a composition comprising anisolated recombinant human N-acetyl-alpha-D-glucosaminidase (rhNaGlu)comprising one or more glycan structures having sufficient amount ofmannose-6-phosphate (M6P) that allows for internalization of the rhNaGluinto a mammalian cell having NaGlu deficiency via M6P receptor-mediatedendocytosis, such that when internalized in vivo, the rhNaGlu restoresat least 50% of NaGlu activity observed in a wild-type cell of the sametype expressing endogenous NaGlu.

In one embodiment, the rhNaGlu protein is N-linked glycosylated. Inanother embodiment, the rhNaGlu protein is O-linked glycosylated. In oneembodiment, the rhNaGlu comprises about 2, 3, 4, 5 or 6 moles of M6P permole of rhNaGlu.

In one embodiment, the rhNaGlu is effectively delivered to the brain ofa mammal having NaGlu deficiency when administered systemically. Inanother embodiment, the rhNaGlu is effectively delivered to the brain ofa mammal having NaGlu deficiency when administered intravenously. Inanother embodiment, the rhNaGlu is effectively delivered to the brain ofa mammal having NaGlu deficiency when administered intrathecally.

In another aspect, the invention provides a transgenic avian comprisinga transgene containing a promoter operably linked to a nucleic acidsequence encoding a recombinant human NaGlu (rhNaGlu), wherein thetransgene is contained in the genome of the transgenic avian andexpressed in an oviduct cell such that the rhNaGlu is glycosylated inthe oviduct cell of the transgenic avian, secreted into lumen of oviductand deposited in egg white of an egg of the transgenic avian.

In one embodiment, the rhNaGlu comprises about 2, 3, 4 or 6 moles of M6Pper mole of rhNaGlu. In another embodiment, the promoter component is anoviduct-specific promoter. In another embodiment, the oviduct-specificpromoter is an ovalbumin promoter. In yet another embodiment, thetransgenic avian is selected from the group consisting of a chicken, aturkey, a duck and a quail.

In another aspect, the invention provides an egg produced by thetransgenic avian of the invention.

In yet another aspect, the invention provides a method of producing arecombinant human NaGlu (rhNaGlu) comprising: a) producing a transgenicavian comprising a transgene having a promoter component operably linkedto a nucleic acid sequence encoding the rhNaGlu set forth in 24-743 ofSEQ ID NO:1, wherein the transgene is contained in the genome of thetransgenic avian and expressed in an oviduct cell, such that the rhNaGluis glycosylated in the oviduct cell of the transgenic avian, secretedinto lumen of oviduct and deposited in egg white of an egg laid by thetransgenic avian; and b) isolating the rhNaGlu from the egg white.

In one embodiment, the promoter component is an oviduct-specificpromoter. In another embodiment, the oviduct-specific promoter is anovalbumin promoter. In one embodiment, the avian is selected from thegroup consisting of a chicken, a turkey, a duck and a quail. In oneembodiment, the avian is chicken.

In another aspect, the invention provides a vector comprising anucleotide sequence encoding a human NaGlu operably linked to anovalbumin promoter. In another aspect, the invention provides a hostcell comprising the vector of the invention. In another aspect, theinvention provides an isolated nucleic acid comprising the nucleic acidsequence of 5232-10248 of SEQ ID NO:4.

In one aspect, the invention provides a pharmaceutical formulationcomprising a composition of the invention in combination with apharmaceutically acceptable carrier, diluent or excipient.

In another aspect, the invention provides a composition comprisingrecombinant human NaGlu protein that crosses the blood brain barrier ofa mammal having NaGlu deficiency when administered intravenously.

In yet another aspect, the invention provides a method of treating asubject suffering from NaGlu deficiency, the method comprisingadministering to the subject a therapeutically effective amount of thecomposition of the invention.

In yet another aspect, the invention provides a method of deliveringrecombinant human NaGlu protein to the brain of a subject suffering fromNaGlu deficiency, the method comprising intravenously administeringrecombinant human NaGlu protein to the subject.

In another aspect, the invention provides a method of transporting arecombinant human NaGlu protein from the circulation across the bloodbrain barrier in a therapeutically effective amount, the methodcomprising intravenously administering a recombinant human NaGlu proteinto a subject having NaGlu deficiency.

In one embodiment, the NaGlu deficiency is Sanfilippo Syndrome B. Inanother embodiment, the subject is a human.

In another embodiment, the recombinant human NaGlu protein isadministered intravenously to the subject at a dosage of about 0.5 toabout 50 mg/kg body weight. In another embodiment, the recombinant humanNaGlu protein is administered intravenously to the subject at a dosageof about 1 to about 30 mg/kg body weight. In another embodiment, therecombinant human NaGlu protein is administered intravenously to thesubject at a dosage of about 6 to about 27 mg/kg body weight.

In yet another embodiment, the recombinant human NaGlu protein isintrathecally administered to the subject. In one embodiment, therecombinant human NaGlu protein is intrathecally administered at adosage of at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 mg/kg bodyweight. In another embodiment, the recombinant human NaGlu protein isintrathecally administered at a dosage of about 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 mg/kg body weight. In another embodiment, the recombinant humanNaGlu protein is administered intrathecally at a dosage of about 10 toabout 30 mg/kg body weight.

In another embodiment, the therapeutically effective amount is an amounteffective to reduce heparan sulfate levels in the brain, the kidney, orthe liver of the subject. In another embodiment, the therapeuticallyeffective amount is an amount effective to increase NaGlu activity inthe brain or the liver of the subject.

In another embodiment, the method further comprises administering asecond therapeutic agent. In one embodiment, the second therapeutic isan immunosuppressant.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the amino acid sequence of human recombinant NaGlu (aminoacid residues 1-23, signal peptide).

FIG. 2 depicts the nucleic acid sequence (cDNA) of human recombinantNaGlu, including the nucleic acid sequence encoding the signal peptide.

FIG. 3 depicts the nucleic acid sequence of 1.1 kb ovalbumin promoter.

FIGS. 4A-D depict the nucleic acid sequence of pSIN-OV-1.1-I-rhNaGluvector used in transgenesis of an avian.

FIG. 5 is a schematic representation of pSIN-OV-1.1-I-rhNaGlu vector.

FIG. 6 depicts Western analysis of rhNaGlu isolated and purified fromegg white of a transgenic Gallus.

FIG. 7 depicts the average concentration of rhNaGlu deposited in eggwhite of transgenic Gallus.

FIG. 8 depicts an oligosaccharide profile of rhNaGlu produced from atransgenic Gallus using HPAEC-PAD.

FIG. 9 depicts uptake analysis of rhNaGlu by human skin fibroblasts (MPSIIIB, NaGlu deficient; Normal, wild-type human skin fibroblast; 1U ofenzymatic activity=nmol of protein/hr).

FIG. 10 depicts uptake inhibition analysis of rhNaGlu (Gallus) usingvarious concentrations of M6P monosaccharide (1U of enzymaticactivity=1μ mol of protein/min).

FIG. 11 depicts a schematic representation of pTT22 vector containing arecombinant human NaGlu fusion construct (AAA-NaGlu: acidic amino acidresidues fused to N-terminus of the full length NaGlu).

FIG. 12 depicts a schematic representation of pTT22 vector containing arecombinant human NaGlu fusion construct (NaGlu-TfRL: transferrinreceptor ligand fused to C-terminus of the full length NaGlu).

FIG. 13 depicts enzymatic activity of AAA-NaGlu produced from HEK293 ascompared to rhNaGlu produced from Gallus.

FIG. 14 depicts enzymatic activity of NaGlu-TfRL produced from HEK293 ascompared to AAA-NaGlu produced from HEK293.

FIG. 15 depicts uptake levels of rhNaGlu (Gallus) into a macrophage cellline (NR8383) over time (48 hours). Cellular NaGlu activity was measuredin units/mg of protein.

FIG. 16 depicts heparan sulfate substrate levels (μg/mg tissue) in thekidney of naglu (^(−/−)) mice following intravenous administration ofvehicle (KO); rhNaGlu gallus at a dosage concentration of 6.25 mg/kg; orrhNaGlu gallus at a dosage concentration of 27 mg/kg. Wild type (WT)mice were untreated.

FIG. 17 depicts heparan sulfate substrate levels (μg/mg tissue) in thebrain of naglu (^(−/−)) mice following intravenous administration ofvehicle (KO); rhNaGlu gallus at a dosage concentration 6.25 mg/kg; orrhNaGlu gallus at a dosage concentration of 27 mg/kg. Wild type (WT)mice were untreated.

FIG. 18 depicts heparan sulfate substrate levels (μg/mg tissue) in theliver of naglu (^(−/−)) mice following intravenous administration ofvehicle (KO); rhNaGlu gallus at a dosage concentration of 6.25 mg/kg; orrhNaGlu gallus at a dosage concentration of 27 mg/kg. Wild type (WT)mice were untreated.

FIG. 19 depicts heparan sulfate substrate levels (μg/mg tissue) in thebrain of naglu (^(−/−)) mice following intrathecal administration ofvehicle (KO) or rhNaGlu gallus at a dosage concentration of 0.31 mg/kg.Wild type (WT) mice were untreated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions comprising recombinant humanNaGlu protein (rhNaGlu) useful for therapy, for example, in thetreatment of NaGlu associated diseases, e.g., Sanfilippo Syndrome B. Thepresent invention is based on a discovery that the rhNaGlu proteindescribed herein contains sufficient amount of oligosaccharides (e.g.,mannose and phosphorylated mannose (i.e., M6P)) to allow efficientcellular uptake via mannose and/or M6P receptor-mediated endocytosis andbe correctly targeted into human cells. Since the rhNaGlu of theinvention is more efficiently taken up into a human cell, the rhNaGlu ofthe invention exhibits increased enzymatic activity as compared to NaGluproteins produced and secreted from unmodified mammalian cells that arenot designed to produce specific glycosylation. Additionally, therhNaGlu described herein has one or more glycosylation patterns thatallow the rhNaGlu to efficiently cross the blood brain barrier (BBB)when administered intravenously. The increased cellular uptake of therhNaGlu protein of the invention minimizes the need for large andfrequent dosing, thereby greatly reducing the potential risk ofimmunogenicity.

Some of the definitions and abbreviations used herein include thefollowing: aa, amino acid(s); bp, base pair(s); CDS, coding sequencecDNA, DNA complementary to an RNA; GalNac, N-acetylgalactosamine; Gal,galactose; GlcNac, N-acetylglucosamine; nt, nucleotide(s); kb, 1,000base pairs; μg, microgram; mL, milliliter; ng, nanogram; and nt,nucleotide.

Certain definitions are set forth herein to illustrate and define themeaning and scope of the various terms used to describe the inventionherein.

The term “avian” as used herein refers to any species, subspecies orstrain of organism of the taxonomic class ava, such as, but not limitedto, chicken, turkey, duck, goose, quail, pheasants, parrots, finches,hawks, crows and ratites including ostrich, emu and cassowary. The termincludes the various known strains of Gallus gallus, or chickens, (forexample, White Leghorn, Brown Leghorn, Barred-Rock, Sussex, NewHampshire, Rhode Island, Ausstralorp, Minorca, Amrox, California Gray,Italian Partridge-colored), as well as strains of turkeys, pheasants,quails, duck, ostriches and other poultry commonly bred in commercialquantities.

The phrases “based on” and “derived from” typically mean obtained from,in whole or in part. For example, a retroviral vector being based on orderived from a particular retrovirus or based on a nucleotide sequenceof a particular retrovirus mean that the genome of the retroviral vectorcontains a substantial portion of the nucleotide sequence of the genomeof the particular retrovirus. The substantial portion can be aparticular gene or nucleotide sequence such as the nucleotide sequenceencoding the gag, pol and/or env proteins or other structural orfunctional nucleotide sequence of the virus genome such as sequencesencoding the long terminal repeats (LTRs) or can be substantially thecomplete retrovirus genome, for example, most (e.g., more than 60% ormore than 70% or more than 80% or more than 90%) or all of theretrovirus genome, as will be apparent from the context in thespecification as the knowledge of one skilled in the art. Examples ofretroviral vectors that are based on or derived from a retrovirus arethe NL retroviral vectors (e.g., NLB) which are derived from the avianleukosis retrovirus (“ALV”) as disclosed in Cosset et al., Journal ofVirology (1991) vol. 65, p 3388-3394.

The term “coding sequence” and “coding region” as used herein refer tonucleotide sequences and nucleic acid sequences, including both RNA andDNA, that encode genetic information for the synthesis of an RNA, aprotein, or any portion of an RNA or protein.

Nucleotide sequences that are not naturally part of a particularorganism's genome or are introduced at a non-native site in theorganism's genome are referred to as “foreign” nucleotide sequences,“heterologous” nucleotide sequences, “recombinant” nucleotide sequencesor “exogenous” nucleotide sequences. In addition, a nucleotide sequencethat has been isolated and then reintroduced into the same type (e.g.,same species) of organism is not considered to be a naturally occurringpart of a particular organism's genome and is therefore consideredexogenous or heterologous. “Heterologous proteins” or “exogenousproteins” can be proteins encoded by foreign, heterologous or exogenousnucleotide sequences and therefore are often not naturally expressed ina cell of the host organism.

As used herein, the terms “exogenous,” “heterologous” and “foreign” withreference to nucleic acids, such as DNA and RNA, are usedinterchangeably and refer to nucleic acid that does not occur naturallyas part of a chromosome, a genome or cell in which it is present orwhich is found in a location(s) and/or in amounts that differ from thelocation(s) and/or amounts in which it occurs in nature. It can benucleic acid that is not endogenous to the genome, chromosome or celland has been exogenously introduced into the genome, chromosome or cell.Examples of heterologous DNA include, but are not limited to, DNA thatencodes a gene product or gene product(s) of interest, for example, forproduction of an encoded protein. Examples of heterologous DNA include,but are not limited to, DNA that encodes traceable marker proteins, DNAthat encodes therapeutic proteins. The terms “heterologous” and“exogenous” can refer to a biomolecule such as a nucleic acid or aprotein which is not normally found in a certain cell, tissue orsubstance produced by an organism or is not normally found in a certaincell, tissue or substance produced by an organism in an amount orlocation the same as that found to occur naturally. For example, aprotein that is heterologous or exogenous to an egg is a protein that isnot normally found in the egg.

The term “construct” as used herein refers to a linear or circularnucleotide sequence such as DNA that has been assembled from more thanone segments of nucleotide sequence which have been isolated from anatural source or have been chemically synthesized, or combinationsthereof.

The term “complementary” as used herein refers to two nucleic acidmolecules that can form specific interactions with one another. In thespecific interactions, an adenine base within one strand of a nucleicacid can form two hydrogen bonds with thymine within a second nucleicacid strand when the two nucleic acid strands are in opposingpolarities. Also in the specific interactions, a guanine base within onestrand of a nucleic acid can form three hydrogen bonds with cytosinewithin a second nucleic acid strand when the two nucleic acid strandsare in opposing polarities. Complementary nucleic acids as referred toherein, can further comprise modified bases wherein a modified adeninemay form hydrogen bonds with a thymine or modified thymine, and amodified cytosine may form hydrogen bonds with a guanine or a modifiedguanine.

The term “expressed” or “expression” as used herein refers to thetranscription of a coding sequence to yield an RNA molecule at leastcomplementary in part to a region of one of the two nucleic acid strandsof the coding sequence. The term “expressed” or “expression” as usedherein can also refer to the translation of an mRNA to produce a proteinor peptide.

The term “expression vector” as used herein refers to a nucleic acidvector that comprises a gene expression controlling region, such as apromoter or promoter component, operably linked to a nucleotide sequenceencoding at least one polypeptide.

The term “fragment” as used herein can refer to, for example, an atleast about 10, 20, 50, 75, 100, 150, 200, 250, 300, 500, 1000, 2000,5000, 6,000, 8,000, 10,000, 20,000, 30,000, 40,000, 50,000 or 60,000nucleotide long portion of a nucleic acid that has been constructedartificially (e.g., by chemical synthesis) or by cleaving a naturalproduct into multiple pieces, using restriction endonucleases ormechanical shearing, or enzymatically, for example, by PCR or any otherpolymerizing technique known in the art, or expressed in a host cell byrecombinant nucleic acid technology known to one of skill in the art.The term “fragment” as used herein can also refer to, for example, an atleast about 5, 10, 15, 20, 25, 30, 40, or 50 amino acid residues lessthan a full length amino acid sequence for NaGlu (i.e., amino acidsequence 24-743 of SEQ ID NO:1), which portion is cleaved from anaturally occurring amino acid sequence by proteolytic cleavage by atleast one protease, or is a portion of the naturally occurring aminoacid sequence synthesized by chemical methods or using recombinant DNAtechnology (e.g., expressed from a portion of the nucleotide sequenceencoding the naturally occurring amino acid sequence) known to one ofskill in the art. “Fragment” may also refer to a portion, for example,of about 50%, about 60%, about 70%, about 80%, about 90%, about 95% orabout 99% of a particular nucleotide sequence or amino acid sequence.

“Functional portion” and “functional fragment” can be usedinterchangeably and as used herein mean a portion or fragment of a wholecapable of performing, in whole or in part, a function of the whole. Forexample, a biologically functional portion of a molecule means a portionof the molecule that performs a biological function of the whole orintact molecule. Functional portions may be of any useful size. Forexample, a functional fragment may range in size from about 20 bases inlength to a length equal to the entire length of the specified sequenceminus one nucleotide. In another example, a functional fragment mayrange in size from about 50 bases in length to a length equal to theentire length of the specified sequence minus one nucleotide. In anotherexample, a functional fragment may range in size from about 50 bases inlength to about 20 kb in length. In another example, a functionalfragment may range in size from about 500 bases in length to about 20 kbin length. In another example, a functional fragment may range in sizefrom about 1 kb in length to about 20 kb in length. In another example,a functional fragment may range in size from about 0.1 kb in length toabout 10 kb in length. In another example, a functional fragment mayrange in size from about 20 bases kb in length to about 10 kb in length.

The term “fully transgenic” or “germline transgenic” refers to an animalsuch as an avian that contains at least one copy of a transgene inessentially all of its cells.

The term “gene expression controlling region” as used herein refers tonucleotide sequences that are associated with a coding sequence andwhich regulate, in whole or in part, expression of the coding sequence,for example, regulate, in whole or in part, the transcription of thecoding sequence. Gene expression controlling regions may be isolatedfrom a naturally occurring source or may be chemically synthesized andcan be incorporated into a nucleic acid vector to enable regulatedtranscription in appropriate cells. The “gene expression controllingregions” may precede, but is not limited to preceding, the region of anucleic acid sequence that is in the region 5′ of the end of a codingsequence that may be transcribed into mRNA.

As used herein, “host cells” refers to cells that harbor vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene.

The term “isolated nucleic acid” as used herein covers, for example, (a)a DNA which has the sequence of part of a naturally occurring genomicmolecule but is not flanked by at least one of the sequences that flankthat part of the molecule in the genome of the species in which itnaturally occurs; (b) a nucleic acid which has been incorporated into avector or into the genomic DNA of a prokaryote or eukaryote in a mannersuch that the resulting vector or genomic DNA is not identical tonaturally occurring DNA from which the nucleic acid was obtained; (c) aseparate molecule such as a cDNA, a genomic fragment, a fragmentproduced by polymerase chain reaction (PCR), ligase chain reaction (LCR)or chemical synthesis, or a restriction fragment; (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein, and (e) a recombinant nucleotide sequence that is partof a hybrid sequence that is not naturally occurring. Isolated nucleicacid molecules of the present invention can include, for example,natural allelic variants as well as nucleic acid molecules modified bynucleotide deletions, insertions, inversions, or substitutions.

The term “nucleic acid” as used herein refers to any linear orsequential array of nucleotides and nucleosides, for example cDNA,genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleosides andderivatives thereof. For ease of discussion, non-naturally occurringnucleic acids may be referred to herein as constructs. Nucleic acids caninclude bacterial plasmid vectors including expression, cloning, cosmidand transformation vectors such as, animal viral vectors such as, butnot limited to, modified adenovirus, influenza virus, polio virus, poxvirus, retroviruses such as avian leukosis virus (ALV) retroviralvector, a murine leukemia virus (MLV) retroviral vector, and alentivirus vector, and the like and fragments thereof. In addition, thenucleic acid can be an LTR of an avian leukosis virus (ALV) retroviralvector, a murine leukemia virus (MLV) retroviral vector, or a lentivirusvector and fragments thereof. Nucleic acids can also include NL vectorssuch as NLB, NLD and NLA and fragments thereof and syntheticoligonucleotides such as chemically synthesized DNA or RNA. Nucleicacids can include modified or derivatized nucleotides and nucleosidessuch as, but not limited to, halogenated nucleotides such as, but notonly, 5-bromouracil, and derivatized nucleotides such as biotin-labelednucleotides.

As used herein, the terms “glycan,” “glycan structure,” “glycan moiety,”“oligosaccharide,” “oligosaccharide structure,” “glycosylation pattern,”“glycosylation profile,” and “glycosylation structure” have essentiallythe same meaning and each refers to one or more structures which areformed from sugar residues and are attached to glycosylated protein suchas human NaGlu. For example, “N-glycan” or “N-linked glycan” refers to aglycan structure attached to a nitrogen of asparagine or arginineside-chain of the glycosylated protein. “O-glycan” or “O-linked glycan”refers to a glycan structure attached to the hydroxyloxygen of serine,threonine, tyrosine, hydroxylysine, or hydroxyproline side chain of theglycosylate protein.

The term “vector” and “nucleic acid vector” as used herein refers to anatural or synthetic single or double stranded plasmid or viral nucleicacid molecule that can be transfected or transformed into cells andreplicate independently of, or within, the host cell genome. A circulardouble stranded vector can be linearized by treatment with anappropriate restriction enzyme based on the nucleotide sequence of thevector. A nucleic acid can be inserted into a vector by cutting thevector with restriction enzymes and ligating the desired piecestogether, as is understood in the art. A typical vector can be comprisedof the following elements operatively linked at appropriate distancesfor allowing functional gene expression: replication origin, promoter,enhancer, 5′ mRNA leader sequence, ribosomal binding site, nucleic acidcassette, termination and polyadenylation sites, and selectable markersequences. One or more of these elements can be omitted in specificapplications. The nucleic acid cassette can include a restriction sitefor insertion of the nucleic acid sequence to be expressed. In afunctional vector the nucleic acid cassette contains the nucleic acidsequence to be expressed including translation initiation andtermination sites. An intron optionally can be included in theconstruct, for example, 5′ to the coding sequence. A vector isconstructed so that the particular coding sequence is located in thevector with the appropriate regulatory sequences, the positioning andorientation of the coding sequence with respect to the control sequencesbeing such that the coding sequence is transcribed under the “control”of the control or regulatory sequences. Modification of the sequencesencoding the particular protein of interest can be desirable to achievethis end. For example, in some cases it can be necessary to modify thesequence so that it can be attached to the control sequences with theappropriate orientation, or to maintain the reading frame. The controlsequences and other regulatory sequences can be ligated to the codingsequence prior to insertion into a vector. Alternatively, the codingsequence can be cloned directly into an expression vector which alreadycontains the control sequences and an appropriate restriction site whichis in reading frame with and under regulatory control of the controlsequences.

The term “operably linked” refers to an arrangement of elements whereinthe components so described are configured so as to perform their usualfunction. Gene expression controlling regions or promoter(s) (e.g.,promoter components) operably linked to a coding sequence are capable ofeffecting the expression of the coding sequence. The controllingsequence(s) or promoter need not be contiguous with the coding sequence,so long as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

“Overexpression”, as used herein, refers to the production of a geneproduct in transgenic organisms that exceeds levels of production innormal or non-transformed organisms.

The term “oviduct” or “oviduct tissue” refers to a tissue of an avianoviduct, such as the magnum, e.g., tubular gland cells, where proteinsare produced with N-linked oligosaccharides that contain increasedamounts of mannose and mammose-6-phosphate (M6P) and substantiallyreduced amounts of galactose and/or sialic acid relative to that ofproteins produced in other tissue of the avian such as liver or kidneytissue.

The term “oviduct-specific promoter” as used herein refers to promotersand promoter components which are functional, i.e., provide fortranscription of a coding sequence, to a large extent, for example,primarily (i.e., more than 50% of the transcription product produced inthe animal by a particular promoter type being produced in oviductcells) or exclusively in oviduct cells of a bird. Examples of oviductspecific promoters include, but are not limited to, ovalbumin promoter,ovomucoid promoter, ovoinhibitor promoter, lysozyme promoter andovotransferrin promoter and functional portions of these promoters,e.g., promoter components. By limiting the expression of NaGlu proteinto the magnum using oviduct specific promoters, deleteriousphysiological effects to the bird as result of expression of theseenzymes in other tissues of the bird can be minimized.

The terms “percent sequence identity,” “percent identity,” “% identity,”“percent sequence homology,” “percent homology,” “% homology” and“percent sequence similarity” can each refer to the degree of sequencematching between two nucleic acid sequences or two amino acid sequences.Such sequence matching can be determined using the algorithm of Karlin &Altschul (1990) Proc. Natl. Acad. Sci. 87: 2264-2268, modified as inKarlin & Altschul (1993) Proc. Natl. Acad. Sci. 90: 5873-5877. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (1990) T. Mol. Biol. Q15: 403-410. BLAST nucleotidesearches are performed with the NBLAST program, score=100,wordlength=12, to obtain nucleotide sequences homologous to a nucleicacid molecule of the invention. BLAST protein searches are performedwith the XBLAST program, score=50, wordlength=3, to obtain amino acidsequences homologous to a reference amino acid sequence. To obtaingapped alignments for comparison purposes, Gapped BLAST is utilized asdescribed in Altschul et al. (1997) Nucl. Acids Res. 25: 3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) are used. Otheralgorithms, programs and default settings may also be suitable such as,but not only, the GCG-Sequence Analysis Package of the U.K. Human GenomeMapping Project Resource Centre that includes programs for nucleotide oramino acid sequence comparisons. A sequence may be at least 50%, 60%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to another sequence, e.g., the NaGlu protein sequenceidentified herein.

The term “avian derived” refers to a composition or substance producedby or obtained from a bird, poultry or avian. “Avian” refers to birdsthat can be kept as livestock, including but not limited to, chickens,duck, turkey, quail and ratites. For example, “avian derived” can referto chicken derived, turkey derived and/or quail derived.

The terms “polynucleotide,” “oligonucleotide”, “nucleotide sequence” and“nucleic acid sequence” can be used interchangeably herein and include,but are not limited to, coding sequences, i.e., polynucleotide(s) ornucleic acid sequence(s) which are transcribed and translated intopolypeptide in vitro or in vivo when placed under the control ofappropriate regulatory or control sequences; controlling sequences,e.g., translational start and stop codons, promoter sequences, ribosomebinding sites, polyadenylation signals, transcription factor bindingsites, transcription termination sequences, upstream and downstreamregulatory domains, enhancers, silencers, DNA sequences to which atranscription factor(s) binds and alters the activity of a gene'spromoter either positively (induction) or negatively (repression) andthe like. No limitations as to length or to synthetic origin aresuggested by the terms described herein.

As used herein the terms “polypeptide” and “protein” refer to a polymerof amino acids, for example, three or more amino acids, in a serialarray, linked through peptide bonds. The term “polypeptide” includesproteins, protein fragments, protein analogues, oligopeptides and thelike. The term “polypeptides” includes polypeptides as defined abovethat are encoded by nucleic acids, produced through recombinanttechnology (e.g., isolated from a transgenic bird), or synthesized. Theterm “polypeptides' further contemplates polypeptides as defined abovethat include chemically modified amino acids or amino acids covalentlyor noncovalently linked to labeling ligands.

The term “promoter” as used herein refers to a DNA sequence useful toinitiate transcription by an RNA polymerase in an avian cell. A“promoter component” is a DNA sequence that can, by itself or incombination with other DNA sequences, effect or facilitatetranscription. Promoter components can be functional fragments ofpromoters.

The terms “recombinant nucleic acid” and “recombinant DNA” as usedherein refer to combinations of at least two nucleic acid sequences thatare not naturally found in a eukaryotic or prokaryotic cell. The nucleicacid sequences may include, but are not limited to, nucleic acidvectors, gene expression regulatory elements, origins of replication,suitable gene sequences that when expressed confer antibioticresistance, protein-encoding sequences and the like. The term“recombinant polypeptide” is meant to include a polypeptide produced byrecombinant DNA techniques such that it is distinct from a naturallyoccurring polypeptide either in its location, purity or structure.Generally, such a recombinant polypeptide will be present in a cell inan amount different from that normally observed in nature.

As used herein, the term “regulatory” sequences or elements includepromoters, enhancers, terminators, stop codons, and other elements thatcan control gene expression.

A “retrovirus”, “retroviral particle,” “transducing particle,” or“transduction particle” refers to a replication-defective orreplication-competent virus capable of transducing non-viral DNA or RNAinto a cell.

A “SIN vector” refers to a self-inactivating vector. In particular, aSIN vector is a retroviral vector having an altered genome such thatupon integration into genomic DNA of the target cell (e.g., avian embryocells), the 5′ LTR of the integrated retroviral vector will not functionas a promoter. For example, a portion or all of the nucleotide sequenceof the retroviral vector that results in the U3 region of the 5′ LTR ofthe retroviral vector once integrated can be deleted or altered in orderto reduce or eliminate promoter activity of the 5′ LTR. In certainexamples, deletion of the CAAT box and/or the TAATA box from U3 of the5′ LTR can result in a SIN vector, as is understood in the art.

The term “sense strand” as used herein refers to a single stranded DNAmolecule from a genomic DNA that can be transcribed into RNA andtranslated into the natural polypeptide product of the gene. The term“antisense strand” as used herein refers to the single strand DNAmolecule of a genomic DNA that is complementary with the sense strand ofthe gene.

A “therapeutic protein” or “pharmaceutical protein” is a substance that,in whole or in part, makes up a drug. In particular, “therapeuticproteins” and “pharmaceutical proteins” include an amino acid sequencewhich in whole or in part makes up a drug.

The terms “promoter,” “transcription regulatory sequence” and “promotercomponent” as used herein refer to nucleotide which regulates thetranscriptional expression of a coding sequence. Exemplary transcriptionregulatory sequences include enhancer elements, hormone responseelements, steroid response elements, negative regulatory elements, andthe like. The “transcription regulatory sequence” can be isolated andincorporated into a vector to enable regulated transcription inappropriate cells of portions of the vector DNA. The “transcriptionregulatory sequence” can precede, but is not limited to, the region of anucleic acid sequence that is in the region 5′ of the end of a proteincoding sequence that is transcribed into mRNA. Transcriptionalregulatory sequence can also be located within a protein coding region,for example, in regions of a gene that are identified as “intron”regions.

The terms “transformation” and “transfection” as used herein refer tothe process of inserting a nucleic acid into a host. Many techniques arewell known to those skilled in the art to facilitate transformation ortransfection of a nucleic acid into a prokaryotic or eukaryoticorganism. These methods involve a variety of techniques, such astreating the cells with certain concentrations of salt, for example, butwithout limitation, a calcium or magnesium salt, or exposing the cellsto an electric field, detergent, or liposome material, to render thehost cell competent for the uptake of the nucleic acid molecules.

As used herein, a “transgenic animal” is any non-human animal, such asan avian species, including the chicken, in which one or more of thecells of the animal contain heterologous nucleic acid introduced by wayof human intervention, such as by transgenic techniques known in the art(see, for example, U.S. patent publication No. 2007/0243165, publishedOct. 18, 2007, the disclosure of which is incorporated in its entiretyherein by reference) including those disclosed herein. The nucleic acidis introduced into an animal, directly or indirectly by introductioninto a cell (e.g., egg or embryo cell) by way of deliberate geneticmanipulation, such as by microinjection or by infection with arecombinant virus. The term genetic manipulation does not includeclassical cross-breeding, or in vitro fertilization, but rather isdirected to the introduction of a recombinant DNA molecule. Thismolecule can be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animal,the transgene can cause cells to express a recombinant form of thetarget protein or polypeptide. The terms “chimeric animal” or “mosaicanimal” are used herein to refer to animals in which a transgene isfound, or in which the recombinant nucleotide sequence is expressed, insome but not all cells of the animal. A germ-line chimeric animalcontains a transgene in its germ cells and can give rise to an offspringtransgenic animal in which most or all cells of the offspring willcontain the transgene.

As used herein, the term “transgene” means a nucleic acid sequence(encoding, for example, a human NaGlu protein) that is partly orentirely heterologous, i.e., foreign, to the animal or cell into whichit is introduced, or, is partly or entirely homologous to an endogenousgene of the transgenic animal or cell into which it is introduced, butwhich is designed to be inserted, or is inserted, into the animal orcell genome in such a way as to alter the genome of the organism intowhich it is inserted (e.g., it is inserted at a location which differsfrom that of the natural gene or its insertion results in a knockout).

As used herein, the term “enzyme replacement therapy (ERT)” refers to atherapeutic strategy for correcting an enzyme deficiency in a subject byadministering the missing enzyme to a subject. For lysosomal enzymereplacement therapy to be effective, the therapeutic enzyme must bedelivered to lysosomes in the appropriate cells in tissues where thestorage defect is manifested. In one embodiment, the enzyme may beadministered to the subject intravenously, intrathecally,intracerebrally, intraventricularly, or intraparenchymaly. In oneembodiment, the enzyme is able to cross the blood brain barrier (BBB).Without intending to be limited by mechanism, it is believed that as theblood perfuses patient tissues, enzyme is taken up by cells andtransported to the lysosome, where the enzyme acts to eliminate materialthat has accumulated in the lysosomes due to the enzyme deficiency.

I. Composition of NaGlu

The present invention provides novel compositions of recombinant humanNaGlu (rhNaGlu or NaGlu) (amino acid sequence 24-743 set forth in SEQ IDNO:1) having patterns of glycosylation that confer an increased cellularuptake and an increased subcellular activity which are particularlyuseful for therapy, for example, in the treatment of Sanfilippo SyndromeB (mucopolysaccharidosis (MPS) IIIB).

In some aspects, the composition can be an isolated mixture of rhNaGlucomprising the amino acid sequence 24-743 of SEQ ID NO:1. In oneembodiment, the mixture contains a sufficient amount of rhNaGlu havingat least one glycan structure that contains phosphorylated mannose(e.g., M6P) or mannose such that the rhNaGlu containing M6P or mannoseis internalized into a human cell deficient in NaGlu and restores atleast 50% of NaGlu activity observed in a wild-type human cell of thesame type that actively expresses endogenous NaGlu. In one aspect, atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93% 94%,95%, 96%, 97%, 98% or 99% of rhNaGlu in the mixture contains at leastone glycan structure having phosphorylated mannose and/or mannose. Inone embodiment, at least 10% of rhNaGlu in the mixture contains at leastone glycan structure having phosphorylated mannose and/or mannose. Inone embodiment, at least 20% of rhNaGlu in the mixture contains at leastone glycan structure having phosphorylated mannose and/or mannose. Inone embodiment, at least 30% of rhNaGlu in the mixture contains at leastone glycan structure having phosphorylated mannose and/or mannose. Inone embodiment, at least 30% of rhNaGlu in the mixture contains at leastone glycan structure having phosphorylated mannose and/or mannose. Inone embodiment, at least 40% of rhNaGlu in the mixture contains at leastone glycan structure having phosphorylated mannose and/or mannose. Inone embodiment, at least 50% of rhNaGlu in the mixture contains at leastone glycan structure having phosphorylated mannose and/or mannose. Inone embodiment, at least 60% of rhNaGlu in the mixture contains at leastone glycan structure having phosphorylated mannose and/or mannose.

In some aspects, the NaGlu contains one or more N-linked glycanstructure. The NaGlu contains at least one phosphorylated mannose (e.g.,M6P or bis-M6P) which allows the protein to be recognized by the Mannose6-phosphate receptor (M6P receptor), and subsequently taken up into ahuman cell, including but not limited to, a skin fibroblast, anendothelial, a neuronal cell, a hepatocyte, a macrophage or any cellthat expresses M6P receptor on the cell surface via M6Preceptor-mediated endocytosis. In one embodiment, the NaGlu contains atleast one mannose (Man). In another embodiment, the NaGlu contains atleast one N-acetylglucosamine (G1cNAc).

In some aspects, the NaGlu contains a glycan structure comprising aphosphorylated mannose (M6P). As used herein, M6P can encompass anyphosphorylated mannose residue and includes mono- and bis-phosphorylatedmannose. In one embodiment, the M6P is present at a concentration thatis about 1, about 2, about 3, about 4, about 5 or about 6 mole(s) permole of protein. In one embodiment, the NaGlu contains M6P at aconcentration that is about 2, about 3, about 4, or about 5 moles permole of protein. In one embodiment, the NaGlu contains M6P at aconcentration that is about 2 moles per mole of protein. In oneembodiment, the NaGlu contains M6P at a concentration that is about 3moles per mole of protein. In one embodiment, the NaGlu contains M6P ata concentration that is about 4 moles per mole of protein. In oneembodiment, the NaGlu contains M6P at a concentration that is about 5moles per mole of protein. In one embodiment, the NaGlu contains M6P ata concentration that is about 6 moles per mole of protein.

In some aspects, the rhNaGlu contains a sufficient amount of M6P forcellular uptake into a human cell having a M6P receptor on the cellsurface via M6P receptor-mediated endocytosis. In one embodiment, asufficient amount of M6P for uptake into a human cell is about 1, 2, 3,4, 5 or 6 moles per mole of protein. The rhNaGlu can be internalizedinto a human cell deficient in NaGlu such that the internalized proteinfully (100% or more) restores a normal level of NaGlu activity in thehuman cell deficient in NaGlu. In one embodiment, the internalizedrhNaGlu protein fully restores a normal level of NaGlu activity in thehuman cell at a concentration that is at least 0.5, 0.6, 0.7, 0.8, 0.9or 1.0 μg/mL. In one embodiment, the internalized protein fully restoresa normal level of NaGlu activity in the human cell deficient in NaGlu ata concentration that is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 μg/mL. Inone embodiment, the internalized protein fully restores a normal levelof NaGlu activity in the human cell at a concentration that is at least20, 30, 40, 50, 60, 70, 80, 90 or 100 μg/mL. As used herein, the normallevel of NaGlu activity is a level of NaGlu activity measured in awild-type human cell of the same type that actively expresses a normalNaGlu enzyme.

In some aspects, the rhNaGlu can be internalized into a human celldeficient in NaGlu such that the protein restores at least about 50%,about 60%, about 70%, about 80%, about 90% or about 95% of NaGluactivity of a normal human cell of the same type. In some embodiments,the rhNaGlu can be internalized into a human cell deficient in NaGlusuch that the internalized rhNaGlu provides a higher enzymatic activitythan that observed in a normal human cell of the same type. In oneembodiment, the rhNaGlu is internalized into a human cell deficient inNaGlu such that the internalized rhNaGlu provides about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9 and about 10-foldhigher activity than that observed in a normal human cell of the sametype. In one embodiment, the rhNaGlu is internalized into a human celldeficient in NaGlu such that the internalized rhNaGlu provides about 15,about 20, about 25, about 30, about 40, about 50, about 60, about 70,about 80, about 90 or about 100-fold higher activity than that observedin a normal human cell.

In one embodiment, the human cell deficient in NaGlu is any human celldeficient in NaGlu that expresses one or more M6P receptors on the cellsurface. In one embodiment, the human cell deficient in NaGlu is a humanmucopolysaccharidosis (MPS) IIIB fibroblast that accumulates heparansulfate. In one embodiment, the human cell deficient in NaGlu is ahepatocyte. In one embodiment, the human cell deficient in NaGlu is aneuronal cell. In one embodiment, the human cell deficient in NaGlu isan endothelial cell. In one embodiment, the human cell deficient inNaGlu is a macrophage.

In some aspects, uptake of rhNaGlu into a human cell is inhibited by thepresence of about 1, about 2, about 3, about 4, about 5, about 6, about7, about 8, about 9 or about 10 mM of competing M6P monosaccharide. Insome aspects, the cellular uptake of rhNaGlu is inhibited by thepresence of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about0.6, about 0.7, about 0.8, about 0.9 or about 1.0 mM of M6Pmonosaccharide. In one embodiment, the cellular uptake of rhNaGlu isinhibited by the presence of about 0.01, about 0.02, about 0.03, about0.04, about 0.05, about 0.06, about 0.07, about 0.08, or about 0.09 mMof M6P monosaccharide.

In some aspects, the rhNaGlu contains mannose in its glycan structuresat a concentration that is about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34 or 35 moles per mole of protein. In oneembodiment, the rhNaGlu has mannose at a concentration that is about 20,21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 moles per mole of protein. TherhNaGlu contains mannose at a concentration that is about 22, 23, 24,25, 26, 27 or 28 moles per mole of protein. The rhNaGlu contains mannoseat a concentration that is about 24 moles per mole of protein. TherhNaGlu protein contains mannose at a concentration that is about 25moles per mole of protein. The rhNaGlu contains mannose at aconcentration that is about 26 moles per mole of protein. The rhNaGlucontains mannose at a concentration that is about 27 moles per mole ofprotein. In one embodiment, the rhNaGlu has mannose at a concentrationthat is between about 20 and about 30 moles per mole of protein.

In some aspects, the rhNaGlu comprises N-acetylglucosamine (G1cNAc). Inone embodiment, the rhNaGlu contains GlcNAc at a concentration that isbetween about 28 and about 42 moles per mole of protein. In oneembodiment, the NaGlu protein has GlcNAc at a concentration that isbetween about 30 and about 40 moles per mole of protein. In oneembodiment, the NaGlu protein comprises GlcNAc at a concentration thatis between about 32 and about 38 moles per mole of protein. In oneembodiment, the NaGlu protein comprises GlcNAc at a concentration thatis between about 34 and about 36 moles per mole of protein. In oneembodiment, the NaGlu protein has GlcNAc at a concentration that isabout 35 moles per mole of protein. In one embodiment, the rhNaGluprotein contains GlcNAc at a concentration that is about 30, 31, 32, 33,34, 35, 36, 37, 38, 39 or 40 moles per mole of protein.

In some aspects, the rhNaGlu contains N-acetylgalactosamine (GalNAc)and/or galactose (Gal). The presence of the GalNAc and Gal typicallyindicates that the NaGlu may contain one or more O-linked glycanstructures which are added to the protein in the Golgi compartment.Accordingly, the present invention optionally includes a compositioncomprising a recombinant human NaGlu that contains one or more O-linkedglycan structure.

In one embodiment, the rhNaGlu contains galactose at a concentrationthat is about 1, 2, 3, 4, 5, 6 or 7 moles per mole of protein. In oneembodiment, the rhNaGlu has galactose at a concentration that is about2, 3, 4, 5 or 6 moles per mole of protein. In one embodiment, therhNaGlu has galactose at a concentration that is about 3 moles per moleof protein. In one embodiment, the rhNaGlu has galactose at aconcentration that is about 4 moles per mole of protein.

In one embodiment, the NaGlu comprises at least one GalNAc molecule permole of protein. In one embodiment, the NaGlu comprises GalNAc at aconcentration that is about 1 or 2 moles per mole of protein.

In one embodiment, the NaGlu contains no fucose. In yet anotherembodiment, the NaGlu contains no glucose. In yet another embodiment,rhNaGlu contains neither fucose nor glucose.

The present invention also contemplates compositions of modified rhNaGluproteins produced from modified nucleic sequences of rhNaGlu. Themodified nucleic acid sequences include deletions, insertions, orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes a functionally equivalent polynucleotide or polypeptide.The encoded protein may also contain deletions, insertions, orsubstitutions of amino acid residues that produce a silent change andresult in a functionally equivalent protein or polypeptide. Deliberateamino acid substitutions can be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the biological activity ofthe NaGlu is retained. For example, negatively charged amino acids caninclude aspartic acid and glutamic acid; positively charged amino acidscan include lysine and arginine; and amino acids with uncharged polarhead groups having similar hydrophilicity values can include leucine,isoleucine, and valine; glycine and alanine; asparagine and glutamine;serine and threonine; phenylalanine and tyrosine.

In other aspects, the rhNaGlu can be modified such that it contains anadditional moiety or second peptide. Although unmodified NaGlu proteinmay cross the blood brain barrier at a high serum concentration,modifications of the protein can be performed to increase the efficiencyof central nervous system (CNS) targeting. In one embodiment,transferrin receptor ligand (TfRL) can be attached to human NaGlu at N-or C-terminus of NaGlu protein. A non-limiting example of TrRL isTHRPPMWSPVWP (SEQ ID NO:5). In one embodiment, the transferrin receptorligand can be attached to human NaGlu C-terminus of the NaGlu protein.In another embodiment, human NaGlu is fused to insulin-like growthfactor receptor (IGF2R) ligand at N- or C-terminus of the NaGlu protein.In yet another embodiment, the NaGlu protein is fused to low densitylipoprotein (LDL) receptor ligand at N- or C-terminus of the NaGluprotein. In one embodiment, the NaGlu protein is fused to a stretch offive to ten consecutive acidic amino acid residues. The acidic aminoacid residues can include aspartic acid (D) or glutamic acid (E).

In one embodiment, the rhNaGlu is produced in a transgenic avian thatcontains a transgene encoding the NaGlu protein. In one embodiment, therhNaGlu is produced in an oviduct cell (e.g., a tubular gland cell) of atransgenic avian (e.g., chicken (Gallus)). In one embodiment, therhNaGlu is glycosylated in the oviduct cell (e.g., tubular gland cell)of the transgenic avian. In one embodiment, the rhNaGlu has aglycosylation pattern resulting from the rhNaGlu being produced in anoviduct cell of a transgenic avian. In one embodiment, the rhNaGlu canbe isolated and purified from the content of the hard shell eggs laid bythe transgenic avian. In one embodiment, the rhNaGlu can be isolated andpurified from egg white of the transgenic avian.

The present invention also includes compositions of an isolated mixtureof NaGlu proteins, such as a mixture of one or more fragments andfull-length rhNaGlu (e.g., 24-743 set forth in SEQ ID NO:1). In oneembodiment, a substantial portion of the mixture contains phosphorylatedM6P. In one embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,85%, 90% 95%, 97%, 98% or 99% of the rhNaGlu in the mixture containsM6P. In yet another embodiment, at least 50% of the isolated rhNaGlu inthe mixture contains M6P. In yet another embodiment, at least 60% of theisolated rhNaGlu in the mixture contains M6P. In yet another embodiment,at least 70% of the isolated rhNaGlu in the mixture contains M6P. In yetanother embodiment, at least 80% of the isolated rhNaGlu in the mixturecontains M6P. In yet another embodiment, at least 90% of the isolatedrhNaGlu in the mixture contains M6P. In yet another embodiment, at least95% of the isolated rhNaGlu in the mixture contains M6P. In yet anotherembodiment, at least 96% of the isolated rhNaGlu in the mixture containsM6P. In yet another embodiment, at least 97% of the isolated rhNaGlu inthe mixture contains M6P. In yet another embodiment, at least 98% of theisolated rhNaGlu in the mixture contains M6P. In yet another embodiment,at least 99% of the isolated rhNaGlu in the mixture contains M6P.

Optionally, the rhNaGlu protein produced from an avian or mammalianexpression system (e.g., CHO, HEK293, or human skin fibroblastcell-line) can be further modified to achieve a favorable glycosylationpattern (i.e., an increased amount of M6P) for cellular uptake whileretaining the biological activity. Additional terminal M6P can beintroduced to the rhNaGlu by the general methods applied to otherhydrolases as described in U.S. Pat. No. 6,679,165, U.S. Pat. No.7,138,262, or U.S. Publication No. 2009/0022702, the entire teachings ofeach of which are incorporated herein by reference. For example, ahighly phosphorylated mannopyranosyl oligosaccharide compound can bederivatized with a chemical compound containing a carbonyl-reactivegroup, followed by oxidizing the rhNaGlu protein to generate carbonyl(aldehyde) group on one glycan structure of the protein, and reactingthe oxidized NaGlu protein with the glycan with the derivatized highlyphosphorylated mannopyranosyl oligosaccharide compound to form a newcompound having hydrazine bond.

II. Vectors

Methods which are well-known to those skilled in the art can be used toconstruct expression vectors containing sequences encoding NaGlu andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed, for example, in Sambrook, J. et al. (1989) Molecular Cloning,A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., andAusubel, F. M. et al. (1989) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., the entire teachings of which areincorporated herein by reference.

A variety of expression vector/host systems can be utilized to expressnucleic acid sequences encoding rhNaGlu. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus) or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or mammalian cellculture systems (e.g., pTT22 vector). Non-limiting examples of the pTT22vector containing human NaGlu cDNA fused to a nucleic acid sequenceencoding acidic amino acid residue and TfRL are shown in FIGS. 11 and12.

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by vertebrate (e.g., avian or mammalian)cells have a signal peptide or secretory leader sequence which iscleaved from the mature protein once export of the growing protein chainacross the rough endoplasmic reticulum (ER) has been initiated. Those ofordinary skill in the art are aware that polypeptides produced in the ERby vertebrate cells generally have a signal peptide fused to theN-terminus of the polypeptide, which is cleaved from the complete or“full length” polypeptide to produce a secreted or “mature” form of thepolypeptide. In certain embodiments, the native signal peptide, e.g.,the MEAVAVAAAVGVLLLAGAGGAAG (1-23 of SEQ ID NO:1) signal peptide ofhuman NaGlu is used, or a functional derivative of that sequence thatretains the ability to direct the secretion of the polypeptide that isoperably associated with it. Alternatively, a heterologous signalpeptide (e.g., a heterologous mammalian or avian signal peptide), or afunctional derivative thereof, may be used. For example, the wild-typeleader sequence may be substituted with the leader sequence of, forexample, human tissue plasminogen activator (tPA) or mouseB-glucuronidase.

The control elements or regulatory sequences can includes thosenon-translated regions of the vector-enhancers, promoters, 5′ and 3′untranslated regions that interact with host cellular proteins to carryout transcription and translation. Such elements can vary in theirstrength and specificity. Depending on the vector system and host cellutilized, any number of suitable transcription and translation elementscan be used. For example, when cloning in bacterial systems, induciblepromoters such as the hybrid lac-Z promoter of the Bluescript™ phagemid(Stratagene, LaJolla, Calif.) or pSport1™ plasmid (Gibco BRL) and thelike can be used. In mammalian cell systems, promoters from mammaliangenes or from mammalian viruses are preferred. If it is necessary togenerate a cell line that contains multiple copies of the sequenceencoding NaGlu, vectors based on SV40 or EBV can be also used with anappropriate selectable marker such as puromycin and ampicillin (see,e.g., FIGS. 11 and 12).

When the rhNaGlu is produced in a transgenic avian, the presentinvention contemplates that the rhNaGlu sequence be placed downstream ofa promoter such that the sequence encoding the rhNaGlu can be expressedin a tissue-specific manner in a transgenic avian. For example, thepromoter can be an oviduct-specific promoter that is largely, but notentirely, specific to the magnum, such as the oviduct-specific promoter,including but not limited to, ovalbumin, lysozyme, conalbumin,ovomucoid, ovomucoid, ovomucin and ovotransferrin promoters. In oneembodiment, the promoter is an ovalbumin promoter, a lysozyme promoter,a conalbumin promoter, an ovomucoid promoter, an ovomucin promoterand/or an ovotransferrin promoter or any functional portion thereof.

Alternatively, a constitutive promoter can be used to express the codingsequence of human NaGlu in an avian. In this case, expression is notlimited to the magnum; expression also occurs in other tissues withinthe avian (e.g., blood). The use of such a transgene, which includes aconstitutive promoter and the coding sequence of NaGlu, is also suitablefor effecting or driving the expression of a protein in the oviduct andthe subsequent secretion of the protein into the egg. In one embodiment,the constitutive promoter can be, for example, a cytomegalovirus (CMV)promoter, a rous-sarcoma virus (RSV) promoter, a murine leukemia virus(MLV) promoter, and β-actin promoter. In one embodiment, the promoter isa CMV promoter, a MDOT promoter, a RSV promoter, a MLV promoter, or amouse mammary tumor virus (MMTV) promoter of any functional portionthereof.

The invention also contemplates any useful fragment or component of thepromoters described herein. The promoter can be at least one segment,fragment or component of a promoter region, such as a segment of theovalbumin, lysozyme, conalbumin, ovomucoid, ovomucin, ovotransferrin,CMV, RSV or MLV promoter region. In a preferred embodiment, the promoteris a segment of the oviduct-specific promoter region which containsessential elements to direct expression of the coding sequence in thetubular gland cells. For example, included in the scope of the presentinvention is a segment, portion or fragment of an oviduct-specificpromoter and/or condensing the critical regulatory elements of theoviduct-specific promoter so that it retains sequences required forexpression in the tubular gland cells of the magnum of the oviduct. Inone embodiment, a segment of the ovalbumin promoter region is used. Thissegment comprises the 5′-flanking region of the ovalbumin gene.

A vector that contains a coding sequence for human NaGlu can be used fortransfecting blastodermal cells of an avian or mammalian cell togenerate stable integrations into the avian or mammalian genome and tocreate a germline transgenic avian or mammalian cell line. Anon-limiting example of such vector is shown in FIGS. 4A-D and 5. In theavian expression system, the human NaGlu coding sequence is operablylinked to a promoter in a positional relationship to express the codingsequence in a transgenic avian, particularly in the tubular gland cellof the magnum of the avian oviduct, such that the recombinant humanNaGlu protein is expressed and deposited in egg white of a hard shellegg laid by the transgenic avian. Additional suitable vectors andmethods to making vectors for expressing rhNaGlu in an avian system arealso disclosed in U.S. Pat. No. 6,730,822; U.S. Pat. No. 6,825,396; U.S.Pat. No. 6,875,588; U.S. Pat. No. 7,294,507; U.S. Pat. No. 7,521,591;U.S. Pat. No. 7,534,929; U.S. Publication No. 2008/0064862A1; and U.S.Patent Publication No. 2006/0185024, the entire teachings of which areincorporated herein by reference. Non-limiting examples of otherpromoters which can be also useful in the present invention include PolIII promoters (for example, type 1, type 2 and type 3 Pol III promoters)such as H1 promoters, U6 promoters, tRNA promoters, RNase MPR promotersand functional portions of each of these promoters. Typically,functional terminator sequences are selected for use in the presentinvention in accordance with the promoter that is employed.

In one embodiment, the vector is a retroviral vector, in which thecoding sequence and the promoter are both positioned between the 5′ and3′ LTRs of the retroviral vector. In one useful embodiment, the LTRs orretroviral vector is derived from an avian leukosis virus (ALV), amurine leukemia virus (MLV) or a lentivirus. One useful retrovirus forrandomly introducing a transgene into the avian genome is thereplication-deficient ALV, the replication-deficient MLV, or thereplication-deficient lentivirus.

The present invention also contemplates the use of self-inactivating(SIN) vectors. SIN vectors can be useful for increasing the quantity ofhuman NaGlu produced in the oviduct of a transgenic avian. This effectcan be further enhanced when the SIN vector does not contain anyselectable marker cassette with a functional promoter (SIN/SC negativevector). In one embodiment, a SIN vector is a retroviral vector havingaltered genome so that the 5′ LTR of the integrated retroviral vectordoes not function as a promoter. In one particular embodiment, a portionor all of the nucleotide sequence of the retroviral vector that resultsin the U3 region of the 5′ LTR of the retroviral vector once integratedcan be deleted or altered in order to reduce or eliminate promoteractivity of the 5′ LTR. A non-limiting example of SIN vector whichcontains an ovalbumin promoter region fused to the coding sequence ofhuman rhNaGlu is shown in FIGS. 4A-D and 5. Functional components of thevector are also tabulated in Table 1.

TABLE 1 Functional components in pSIN-OV-1.1 kb-I-rhNaGlu Functionalcomponents Nucleotide Sequence in SEQ ID NO: 4 poly A site 634-639Partial gag 692-945 LTR (RAV2) 1243-1588 Partial LTR (RAV2) 4691-4863ALV CTE 4899-4986 1.1 kb Ovalbumin promoter 5232-6363 DHS II 5334-5714DHS I 6064-6364 Exon L 6364-6410 Intron 1 6411-7999 NaGlu  8017-10248

Any of the vectors described herein can include a sequence encoding asignal peptide that directs secretion of the protein expressed by thevector's coding sequence from, for example, the tubular gland cells ofthe oviduct of an avian. Where a recombinant human NaGlu protein wouldnot otherwise be secreted, the vector containing the coding sequence ismodified to comprise a DNA sequence comprising about 60 bp encoding asignal peptide from, for example, the lysozyme gene. The DNA sequenceencoding the signal peptide is inserted in the vector such that it islocated at the N-terminus of the rhNaGlu protein encoded by the DNA.

Further, the coding sequences of vectors used in any of the methods ofthe present invention can be provided with a 3′ untranslated region (3′UTR) to confer stability to the RNA produced. When a 3′ UTR is added toa retroviral vector, the orientation of the promoter, the codingsequence and the 3′ UTR is preferably reversed with respect to thedirection of the 3′ UTR, so that the addition of the 3′ UTR does notinterfere with transcription of the full-length genomic RNA. In oneembodiment, the 3′ UTR may be that of the ovalbumin gene, lysozyme geneor any 3′ UTR that is functional in a magnum cell, i.e., the SV40 lateregion.

III. Transgenic Avians

Transgenes described herein can be introduced into avian embryonicblastodermal cells to produce a transgenic chicken, transgenic turkey,transgenic quail and other avian species that carry the transgeneencoding recombinant human NaGlu in the genome of its germ-line tissue.In one aspect of the invention, a transgenic avian that produces rhNaGluis created by transduction of embryonic blastodermal cells withreplication-defective or replication-competent retroviral particlescarrying the transgene between the 5′ and 3′ LTRs of the retroviralvector. For instance, an avian leukosis virus (ALV) retroviral vector ora murine leukemia virus (MLV) retroviral vector can be used. An RNA copyof the modified retroviral vector packaged into viral particles can beused to infect embryonic blastoderms which develop into transgenicavians.

By the methods of the present invention, transgenes can be introducedinto embryonic blastodermal cells of various avian species. For example,the methods can be applied to produce a transgenic chicken, transgenicturkey, transgenic quail, transgenic duct, and other avian species, thatcarry the transgene in the genome of its germ-line tissue in order toproduce proteins of the invention. The blastodermal cells are typicallystage VII-XII cells as defined by Eyal-Giladi and Kochav (1976), or theequivalent thereof. In a preferred embodiment, the blastoderm cells areat or near stage X.

In one method of transfecting blastodermal cells, a packagedretroviral-based vector can be used to deliver the vector into embryonicblastodermal cells so that the vector is integrated into the aviangenome. Such viral particles (i.e., transduction particles) are producedfor the vector and titered to determine the appropriate concentrationthat can be used to inject embryos. In one embodiment, avian eggs arewindowed according to the procedure described in U.S. Pat. No.5,897,998, the disclosure of which is incorporated herein by referencein its entirety, and the eggs are injected with transducing particles ator near stage X.

The transgenic avians of the invention which produce rhNaGlu aredeveloped from the blastodermal cells into which the vector has beenintroduced. The resulting embryo is allowed to develop and the chickallowed to mature. At this stage, the transgenic avian produced fromblastodermal cells is known as a founder and is chimeric with respect tothe cells carrying the transgene and is referred to G0. G0 founderavians are typically chimeric for each inserted transgene. That is, onlysome of the cells of the G0 transgenic bird contain the transgene. Somefounders carry the transgene in tubular gland cells in the magnum oftheir oviducts. These avians express the rhNaGlu protein encoded by thetransgene in their oviducts. The NaGlu protein may also be expressed inother tissues (e.g., blood) in addition to the oviduct. Some foundersare germ-line founders that carry the transgene in the genome of thegerm-line tissues, and may also carry the transgene in oviduct magnumtubular gland cells that express the exogenous protein.

The transgenic avian can carry the transgene in its germ-line providingtransmission of the exogenous transgene to the avian's offspring stablyin a Mendelian fashion. The G0 generation is typically hemizygous forthe transgene encoding rhNaGlu. The G0 generation can be bred tonon-transgenic animals to give rise to G1 transgenic offspring which arealso hemizygous for the transgene and contain the transgene inessentially all of the bird's cells. The G1 hemizygous offspring can bebred to non-transgenic animals giving rise to G2 hemizygous offspring ormay be bred together to give rise to G2 offspring homozygous for thetransgene. Substantially all of the cells of avians which are positivefor the transgene that are derived from G1 offspring contain thetransgene. In one embodiment, hemizygotic G2 offspring from the sameline can be bred to produce G3 offspring homozygous for the transgene.In another embodiment, hemizygous G0 or G1 animals, for example, arebred together to give rise to homozygous G1 offspring containing twocopies of the transgene(s) in each cell of the animal. These are merelyexamples of certain useful breeding methods and the present inventioncontemplates the employment of any useful breeding method such as thoseknown to individuals of ordinary skill in the art.

IV. Production of rhNaGlu

The rhNaGlu can be produced using a transgenic avian that contains inthe genome a transgene encoding rhNaGlu. In one embodiment, thetransgenic avian is a germline transgenic chicken, quail, duck orturkey. In one particularly useful embodiment, the invention is drawn tothe production of NaGlu which can be produced in the oviduct of achicken.

Production of rhNaGlu with or without modification in the avian system(e.g., in the avian oviduct) is within the scope of the invention. Inone embodiment, the unmodified rhNaGlu comprises the wild-type aminoacid sequence (24-743 of SEQ ID NO:1) with a glycosylation structure(i.e., M6P) that enables efficient uptake by human cells. In anotherembodiment, the modified protein can be an rhNaGlu fusion protein havinga glycosylation pattern (i.e., M6P) that enables efficient uptake byhuman cells.

A suitable avian vector that contains a nucleic acid sequence encoding aNaGlu protein, operably linked to a tissue-specific or constitutivepromoter that drives expression of the encoding sequence in the chickenoviduct are introduced into chicken embryonic cells at or near stage Xas described herein. The transformed embryonic cells are incubated underconditions conducive to hatching live chicks. Live chicks are nurturedinto a mature chimeric chicken which are mated with a non-transgenicchicken naturally or via artificial insemination. A transgenic chickenis identified by screening progeny for germline incorporation of theprotein encoding sequence. The transgenic progeny can be mated withanother transgenic or a non-transgenic chicken to produce a fullygermline transgenic hen that lays eggs.

The rhNaGlu can be produced in a tissue-specific manner. For example,rhNaGlu can be expressed in the oviduct, blood and/or other cells ortissues of the transgenic avian. In one embodiment, the NaGlu isexpressed in the tubular gland cells of the magnum of the oviduct of thetransgenic avian, secreted into the lumen of the oviduct, and depositedinto egg white. In one embodiment, egg white containing rhNaGlu isharvested and stored in bulk at a temperature ranging from 4° C. to −20°C. The NaGlu is then isolated and purified from the contents of the eggsusing various methods known in the art.

One aspect of the present invention relates to avian hard shell eggs(e.g., chicken hard shell eggs) which contain the rhNaGlu protein. TherhNaGlu produced and secreted by the transgenic avian is glycosylated ina manner favorable to cellular uptake by a human cell. The protein maybe present in any useful amount. In one embodiment, the protein ispresent in an amount in a range between about 0.01 μg per hard-shell eggand about 1 gram per hard-shell egg. In another embodiment, the proteinis present in an amount in a range of between about 1 μg per hard-shellegg and about 1 gram per hard-shell egg. For example, the protein may bepresent in an amount in a range of between about 10 μg per hard-shellegg and about 1 gram per hard-shell egg (e.g., a range of between about10 μg per hard-shell egg and about 400 milligrams per hard-shell egg).

In one embodiment, the rhNaGlu is present in the egg white of the egg.In one embodiment, the rhNaGlu is present in an amount in a range ofbetween about 1 ng per milliliter of egg white and about 0.2 gram permilliliter of egg white. For example, the rhNaGlu may be present in anamount in a range of between about 0.1 μg per milliliter of egg whiteand about 0.2 gram per milliliter of egg white (e.g., the rhNaGlu may bepresent in an amount in a range of between about 1 μg per milliliter ofegg white and about 100 milligrams per milliliter of egg white. In oneembodiment, the rhNaGlu is present in an amount in a range of betweenabout 1 μg per milliliter of egg white and about 50 milligrams permilliliter of egg white. For example, the rhNaGlu may be present in anamount in a range of about 1 μg per milliliter of egg white and about 10milligrams per milliliter of egg white (e.g., the rhNaGlu may be presentin an amount in a range of between about 1 μg per milliliter of eggwhite and about 1 milligrams per milliliter of egg white). In oneembodiment, the rhNaGlu is present in an amount of more than 0.1 μg permilliliter of egg white. In one embodiment, the rhNaGlu is present in anamount of more than 0.5 μg per milliliter of egg white. In oneembodiment, the rhNaGlu is present in an amount of more than 1 μg permilliliter of egg white. In one embodiment, the protein is present in anamount of more than 1.5 μg per milliliter of egg white. In oneembodiment, the rhNaGlu is present in an amount of more than 0.5 μg permilliliter of egg white. In one embodiment, the protein is present in anamount of more than 0.1 μg per milliliter of egg white.

In one embodiment, the rhNaGlu is present in an amount of 20 mg/L, 30mg/L, 40 mg/L, 50 mg/L, 60 mg/L, 70 mg/L, 80 mg/L, 90 mg/L, 100 mg/L,120 mg/L, 130 mg/L, 140 mg/L, 150 mg/L, 160 mg/L, 170 mg/L, 200 mg/L,300 mg/L, 400 mg/L, 500 mg/L, 600 mg/L, 700 mg/L, 800 mg/L, 900 mg/L, or1,000 mg/L egg white. In one embodiment, the rhNaGlu is present in anamount of about 100 mg/L of egg white. In one embodiment, the rhNaGlu ispresent in an amount of about 200 mg/L of egg white.

V. Host Cells

The present invention also contemplates rhNaGlu produced in any usefulprotein expression system including, without limitation, cell culture(e.g., avian cells, CHO cells, HEK293 cells and COS cells), yeast,bacteria, and plants.

A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed NaGluin the desired fashion. Such modifications of the polypeptide of NaGluinclude, without limitation, glycosylation, phosphorylation, orlipidation. Different host cells such as CHO, COS, HeLa, MDCK, HEK293and W138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, can be chosen toensure the correct modification and processing of the fusion protein ofthe present invention. An avian tumor cell line is also contemplated asa host cell for expressing the polypeptide of the present invention.Examples of a useful avian cell line (e.g., an avian oviduct tumor cellline) are described in U.S. Pat. Publication No. 2009/0253176, theentire teachings of which are incorporated herein by reference.

VI. Pharmaceutical Compositions

The present invention also features pharmaceutical compositionscomprising isolated and substantially purified rhNaGlu or apharmaceutically acceptable salt thereof. The recombinant human NaGluproteins may be administered using one or more carriers, e.g., as partof a pharmaceutical formulation, or without a carrier. The carrier(s)must be “acceptable” in the sense of being compatible with the otheringredients of the formulation and not deleterious to the recipientthereof. Compositions comprising such carriers, including compositemolecules, are formulated by well-known conventional methods (see, forexample, Remington's Pharmaceutical Sciences, 14th Ed., Mack PublishingCo., Easton, Pa.), the entire teachings of which are incorporated hereinby reference. The carrier may comprise a diluent. In one embodiment, thepharmaceutical carrier can be a liquid and the protein may be in theform of a solution. The pharmaceutical carrier can be wax, fat, oralcohol. In another embodiment, the pharmaceutically acceptable carriermay be a solid in the form of a powder, a lyophilized powder, or atablet. In one embodiment, the carrier may comprise a liposome or amicrocapsule.

In some embodiments, a pharmaceutical composition comprising recombinanthuman NaGlu protein further comprises a buffer. Exemplary buffersinclude acetate, phosphate, citrate and glutamate buffers. Exemplarybuffers also include lithium citrate, sodium citrate, potassium citrate,calcium citrate, lithium lactate, sodium lactate, potassium lactate,calcium lactate, lithium phosphate, sodium phosphate, potassiumphosphate, calcium phosphate, lithium maleate, sodium maleate, potassiummaleate, calcium maleate, lithium tartarate, sodium tartarate, potassiumtartarate, calcium tartarate, lithium succinate, sodium succinate,potassium succinate, calcium succinate, lithium acetate, sodium acetate,potassium acetate, calcium acetate, and mixtures thereof. In someembodiments, the buffer is trisodium citrate dihydrate. In someembodiments, the buffer is citric acid monohydrate. In some embodiments,a pharmaceutical composition comprises trisodium citrate dehydrate andcitric acid monohydrate.

In some embodiments, a pharmaceutical composition comprising recombinanthuman NaGlu protein further comprises a stabilizer. Exemplarystabilizers include albumin, trehalose, sugars, amino acids, polyols,cyclodextrins, salts such as sodium chloride, magnesium chloride, andcalcium chloride, lyoprotectants, and mixtures thereof. In someembodiments, a pharmaceutical composition comprises human serum albumin

In some embodiments, it is desirable to add a surfactant to thepharmaceutical composition. Exemplary surfactants include nonionicsurfactants such as Polysorbates (e.g., Polysorbates 20 or 80);poloxamers (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS);sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-,linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g., Pluronics, PF68, etc). Typically,the amount of surfactant added is such that it reduces aggregation ofthe protein and minimizes the formation of particulates oreffervescences. For example, a surfactant may be present in aformulation at a concentration from about 0.001-0.5% (e.g., about0.005-0.05%, or 0.005-0.01%). In particular, a surfactant may be presentin a formulation at a concentration of approximately 0.005%, 0.01%,0.02%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%, etc. Ranges and valuesintermediate to the above recited ranges and values are alsocontemplated to be part of the invention.

In some embodiments, suitable pharmaceutical compositions of theinvention may further include one or more bulking agents, in particular,for lyophilized formulations. A “bulking agent” is a compound which addsmass to the lyophilized mixture and contributes to the physicalstructure of the lyophilized cake. For example, a bulking agent mayimprove the appearance of lyophilized cake (e.g., essentially uniformlyophilized cake). Suitable bulking agents include, but are not limitedto, sodium chloride, lactose, mannitol, glycine, sucrose, trehalose,hydroxyethyl starch. Exemplary concentrations of bulking agents are fromabout 1% to about 10% (e.g., 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%,4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, and10.0%). Ranges and values intermediate to the above recited ranges andvalues are also contemplated to be part of the invention. Thepharmaceutical compositions can be in the form of a sterile lyophilizedpowder for injection upon reconstitution with a diluent. The diluent canbe water for injection, bacteriostatic water for injection, or sterilesaline. The lyophilized powder may be produced by freeze drying asolution of the fusion protein to produce the protein in dry form. As isknown in the art, the lyophilized protein generally has increasedstability and a longer shelf-life than a liquid solution of the protein.

Pharmaceutical formulations include those suitable for oral, rectal,nasal, topical (including buccal and sub-lingual), vaginal or parenteraladministration. Preferably, the pharmaceutical formulations of theinvention include those suitable for administration by injectionincluding intrathecal, intraparenchymal, intracerebral,intraventricular, intramuscular, sub-cutaneous and intravenousadministration. In one embodiment, the formulations of the invention aresuitable for intravenous administration. In another embodiment, theformulations of the invention are suitable for intrathecaladministration. The pharmaceutical formulations of the invention alsoinclude those suitable for administration by inhalation or insufflation.The formulations can, where appropriate, be conveniently presented indiscrete dosage units and can be prepared by any of the methods wellknown in the art of pharmacy. The methods of producing thepharmaceutical formulations typically include the step of bringing thetherapeutic proteins into association with liquid carriers or finelydivided solid carriers or both and then, if necessary, shaping theproduct into the desired formulation.

Recombinant human NaGlu proteins of the invention can also be formulatedfor parenteral administration (e.g., by injection, for example bolusinjection or continuous infusion) and can be presented in unit dose formin ampoules, pre-filled syringes, small volume infusion or in multi-dosecontainers with an added preservative. The therapeutic proteins can beinjected by, for example, subcutaneous injections, intramuscularinjections, intrathecal injections, intracerebral injections,intraparenchymal injections, intraventricular injections, andintravenous (IV) infusions or injections.

In one embodiment, the recombinant human NaGlu protein is administeredintravenously by IV infusion by any useful method. In one example, therecombinant human NaGlu protein can be administered by intravenousinfusion through a peripheral line. In another example, the recombinanthuman NaGlu protein can be administered by intravenous infusion througha peripherally inserted central catheter. In another example, therecombinant human NaGlu protein can be administered by intravenousinfusion facilitated by an ambulatory infusion machine attached to avenous vascular access port. In one embodiment of intravenous infusion,the medication is administered over a period of 1 to 8 hours dependingon the amount of medication to be infused and the patient's previousinfusion-related reaction history, as determined by a physician skilledin the art. In another embodiment, the recombinant human NaGlu proteinis administered intravenously by IV injection. In another embodiment,the recombinant human NaGlu protein can be administered viaintraperitoneal or intrathecal injection.

In some embodiments, the therapeutic proteins are administered byinfusion, and the infusion can occur over an extended time period, forexample, 30 minutes to 10 hours. Thus, the infusion can occur, forexample, over a period of about 1 hour, about 2 hours, about 3 hours,about 4 hours, or about 5 hours. The infusion can also occur at variousrates. Thus, for example, the infusion rate can be about 1 mL per hourto about 20 mL per hour. In some embodiments, the infusion rate is 5 mLto 10 mL per hour. In one embodiment, the infusion rate is 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mL per hour.In one embodiment, the infusion rate is 0.1 to 5 mg/kg/hr. In oneembodiment, the infusion rate is about 0.1, about 0.2, about 0.3, about0.5, about 1.0, about 1.5, about 2.0, or about 3 mg/kg/hr. Ranges andvalues intermediate to the above recited ranges and values are alsocontemplated to be part of the invention.

The therapeutic proteins can take such forms as suspensions, solutions,or emulsions in oily or aqueous vehicles, and can contain formulatoryagents such as suspending, stabilizing and/or dispersing agents. Therecombinant human NaGlu proteins can be in powder form, obtained byaseptic isolation of sterile solid or by lyophilization from solution,for constitution with a suitable vehicle, e.g., sterile, pyrogen-freewater, before use.

Formulations in accordance with the present invention can be assessedbased on product quality analysis, reconstitution time (if lyophilized),quality of reconstitution (if lyophilized), high molecular weight,moisture, and glass transition temperature. Typically, protein qualityand product analysis include product degradation rate analysis usingmethods including, but not limited to, size exclusion HPLC (SE-HPLC),cation exchange-HPLC (CEX-HPLC), X-ray diffraction (XRD), modulateddifferential scanning calorimetry (mDSC), reversed phase HPLC(RP-HPLC),multi-angle light scattering (MALS), fluorescence, ultravioletabsorption, nephelometry, capillary electrophoresis (CE), SDS-PAGE, andcombinations thereof. In some embodiments, evaluation of product inaccordance with the present invention may include a step of evaluatingappearance (either liquid or cake appearance).

Generally, formulations (lyophilized or aqueous) can be stored forextended periods of time at room temperature. Storage temperature maytypically range from 0° C. to 45° C. (e.g., 4° C., 20° C., 25° C. or 45°C.). Formulations may be stored for a period of months to a period ofyears. Storage time generally will be 24 months, 12 months, 6 months,4.5 months, 3 months, 2 months or 1 month. Formulations can be storeddirectly in the container used for administration, eliminating transfersteps. Ranges and values intermediate to the above recited ranges andvalues are also contemplated to be part of the invention.

Formulations can be stored directly in the lyophilization container (iflyophilized), which may also function as the reconstitution vessel,eliminating transfer steps. Alternatively, lyophilized productformulations may be measured into smaller increments for storage.Storage should generally avoid circumstances that lead to degradation ofthe proteins, including but not limited to exposure to sunlight, UVradiation, other forms of electromagnetic radiation, excessive heat orcold, rapid thermal shock, and mechanical shock. The pharmaceuticalcompositions according to the invention can also contain other activeingredients such as immunosuppressive agents, antimicrobial agents, orpreservatives, discussed in more detail below.

VII. Methods of Treatment

The present invention also provides methods of treating NaGlu-associateddiseases, e.g., Sanfilippo Syndrome B. Recombinant NaGlu employed inaccordance with the invention includes recombinant NaGlu which can beproduced in any useful protein expression system including, withoutlimitation, cell culture (e.g., CHO cells, COS cells), bacteria such asE. coli, transgenic animals such as mammals and avians (e.g., chickens,duck, and turkey) and in plant systems (e.g., duck weed and tobaccoplants). In one embodiment, the recombinant NaGlu is produced in atransgenic animal, such as an avian.

In one embodiment, the method comprises administering to the subject arecombinant human NaGlu protein (rhNaGlu), for instance a recombinanthuman NaGlu protein containing a sufficient amount of oligosaccharides(e.g., mannose and phosphorylated mannose (i.e., M6P)), in an amountsufficient to treat (e.g., reduce, ameliorate) or prevent one or moresymptoms of a NaGlu deficiency or NaGlu associated disease. Therecombinant human NaGlu protein can be administered therapeutically orprophylactically, or both. The recombinant human NaGlu protein (rhNaGlu)can be administered to the subject, alone or in combination with othertherapeutic modalities as described herein.

The terms “treat,” “treating,” and “treatment” refer to methods ofalleviating, abating, or ameliorating a disease or symptom, preventingan additional symptom, ameliorating or preventing an underlying cause ofa symptom, inhibiting a disease or condition, arresting the developmentof a disease or condition, relieving a disease or condition, causingregression of a disease or condition, relieving a condition caused bythe disease or condition, or stopping a symptom of the disease orcondition either prophylactically and/or after the symptom has occurred.

“Therapeutically effective dose” as used herein refers to the dose(e.g., amount and/or interval) of drug required to produce an intendedtherapeutic response (e.g., reduction of heparan sulfate levels and/orincrease in NaGlu activity in a target tissue). A therapeuticallyeffective dose refers to a dose that, as compared to a correspondingsubject who has not received such a dose, results in improved treatment,healing, prevention, or amelioration of a disease, disorder, or sideeffect, or a decrease in the rate of the occurrence or advancement of adisease or disorder. The term also includes within its scope, doseseffective to enhance physiological functions.

As used herein, the term “subject” or “patient” is intended to includehuman and non-human animals. Non-human animals include all vertebrates,e.g., mammals and non-mammals, such as non-human primates, sheep, dogs,cats, cows, horses, chickens, amphibians, and reptiles. Preferredsubjects include human subjects having a NaGlu deficiency or NaGluassociated disease.

As used herein a “NaGlu associated disease” is a disease or conditionwhich is mediated by NaGlu activity or is associated with aberrant NaGluexpression or activity. An example of an NaGlu associated diseaseincludes, but is not limited to, NaGlu deficiency such as SanflippoSyndrome B (also known as mucopolysaccharidosis type IIIB)

The therapeutic methods of the present invention encompass any route ofadministration which facilitates the uptake or transport of therecombinant human NaGlu protein into the pertinent organs and tissues.In one embodiment, the methods of the invention include delivering therecombinant human NaGlu proteins of the invention to the CNS (centralnervous system), the kidney, or the liver of a subject for the treatmentof a NaGlu associated disease (e.g., NaGlu deficiency). For example, therecombinant human NaGlu protein may be administered to the patientintravenously (e.g., via intravenous injection or intravenous infusion)and surprisingly crosses the blood brain barrier (BBB) of the subjecthaving NaGlu deficiency. In another embodiment of the invention, therecombinant human NaGlu protein is administered to the patientintrathecally.

A. Device for Intrathecal Delivery

Various devices may be used for intrathecal delivery according to thepresent invention. In some embodiments, a device for intrathecaladministration contains a fluid access port (e.g., injectable port); ahollow body (e.g., catheter) having a first flow orifice in fluidcommunication with the fluid access port and a second flow orificeconfigured for insertion into spinal cord; and a securing mechanism forsecuring the insertion of the hollow body in the spinal cord. As anon-limiting example, a suitable securing mechanism contains one or morenobs mounted on the surface of the hollow body and a sutured ringadjustable over the one or more nobs to prevent the hollow body (e.g.,catheter) from slipping out of the spinal cord. In various embodiments,the fluid access port comprises a reservoir. In some embodiments, thefluid access port comprises a mechanical pump (e.g., an infusion pump).In some embodiments, an implanted catheter is connected to either areservoir (e.g., for bolus delivery), or an infusion pump. The fluidaccess port may be implanted or external.

In some embodiments, intrathecal administration may be performed byeither lumbar puncture (i.e., slow bolus) or via a port-catheterdelivery system (i.e., infusion or bolus). In some embodiments, thecatheter is inserted between the laminae of the lumbar vertebrae and thetip is threaded up the thecal space to the desired level (generallyL3-L4).

Relative to intravenous administration, a single dose volume suitablefor intrathecal administration is typically small. Typically,intrathecal delivery according to the present invention maintains thebalance of the composition of the CSF as well as the intracranialpressure of the subject. In some embodiments, intrathecal delivery isperformed absent the corresponding removal of CSF from a subject. Insome embodiments, a suitable single dose volume may be e.g., less thanabout 10 mL, 8 mL, 6 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1.5 mL, 1 mL, or 0.5mL. In some embodiments, a suitable single dose volume may be about0.5-5 mL, 0.5-4 mL, 0.5-3 mL, 0.5-2 mL, 0.5-1 mL, 1-3 mL, 1-5 mL, 1.5-3mL, 1-4 mL, or 0.5-1.5 mL. In some embodiments, intrathecal deliveryaccording to the present invention involves a step of removing a desiredamount of CSF first. In some embodiments, less than about 10 mL (e.g.,less than about 9 mL, 8 mL, 7 mL, 6 mL, 5 mL, 4 mL, 3 mL, 2 mL, 1 mL) ofCSF is first removed before intrathecal administration. In those cases,a suitable single dose volume may be e.g., more than about 3 mL, 4 mL, 5mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, or 20 mL. Ranges and valuesintermediate to the above recited ranges and values are alsocontemplated to be part of the invention.

Various other devices may be used to effect intrathecal administrationof a therapeutic composition. For example, formulations containingdesired enzymes may be given using an Ommaya reservoir which is incommon use for intrathecally administering drugs for meningealcarcinomatosis (Lancet 2: 983-84, 1963). More specifically, in thismethod, a ventricular tube is inserted through a hole formed in theanterior horn and is connected to an Ommaya reservoir installed underthe scalp, and the reservoir is subcutaneously punctured tointrathecally deliver the particular enzyme being replaced, which isinjected into the reservoir. Other devices for intrathecaladministration of therapeutic compositions or formulations to anindividual are described in U.S. Pat. No. 6,217,552, the entire contentsof which, as they relate to these devices, are incorporated herein byreference. Alternatively, the drug may be intrathecally given, forexample, by a single injection, or continuous infusion. It should beunderstood that the dosage treatment may be in the form of a single doseadministration or multiple doses.

For injection, formulations of the invention can be formulated in liquidsolutions. In addition, the NaGlu enzyme may be formulated in solid formand re-dissolved or suspended immediately prior to use. Lyophilizedforms are also included. The injection can be, for example, in the formof a bolus injection or continuous infusion (e.g., using infusion pumps)of the NaGlu enzyme.

In one embodiment of the invention, the NaGlu enzyme is administered bylateral cerebro ventricular injection into the brain of a subject. Theinjection can be made, for example, through a burr hole made in thesubject's skull. In another embodiment, the enzyme and/or otherpharmaceutical formulation is administered through a surgically insertedshunt into the cerebral ventricle of a subject. For example, theinjection can be made into the lateral ventricles, which are larger. Insome embodiments, injection into the third and fourth smaller ventriclescan also be made.

In yet another embodiment, the pharmaceutical compositions used in thepresent invention are administered by injection into the cisterna magna,or lumbar area of a subject.

In another embodiment of the method of the invention, thepharmaceutically acceptable formulation provides sustained delivery,e.g., “slow release” of the enzyme or other pharmaceutical compositionused in the present invention, to a subject for at least one, two,three, four weeks or longer periods of time after the pharmaceuticallyacceptable formulation is administered to the subject.

As used herein, the term “sustained delivery” refers to continualdelivery of a pharmaceutical formulation of the invention in vivo over aperiod of time following administration, preferably at least severaldays, a week or several weeks. Sustained delivery of the composition canbe demonstrated by, for example, the continued therapeutic effect of theenzyme over time (e.g., sustained delivery of the enzyme can bedemonstrated by continued reduced amount of storage granules in thesubject). Alternatively, sustained delivery of the enzyme may bedemonstrated by detecting the presence of the enzyme in vivo over time.

B. Intravenous Delivery

As discussed above, one of the surprising features of the presentinvention is that the recombinant human NaGlu proteins of the inventionare able to effectively and extensively diffuse across the blood brainbarrier (BBB) and brain surface and penetrate various layers or regionsof the brain, including deep brain regions, when administeredintravenously. The methods of the present invention effectively deliverthe rhNaGlu proteins to various tissues, neurons or cells of the centralnervous system (CNS), which are hard to target by existing CNS deliverymethods. Furthermore, the methods of the present invention deliversufficient amounts of the recombinant human NaGlu proteins to the bloodstream and various peripheral organs and tissues.

“Intravenous injection,” often medically referred to as IV push or bolusinjection, refers to a route of administration in which a syringe isconnected to the IV access device and the medication is injecteddirectly, typically rapidly and occasionally up to a period of 15minutes if it might cause irritation of the vein or a too-rapid effect.Once a medicine has been injected into the fluid stream of the IVtubing, there must be some means of ensuring that it gets from thetubing to the patient. Usually this is accomplished by allowing thefluid stream to flow normally and thereby carry the medicine into thebloodstream. However, in some cases a second fluid injection, sometimescalled a “flush,” is used following the first injection to facilitatethe entering of the medicine into the bloodstream.

“Intravenous infusion” refers to a route of administration in whichmedication is delivered over an extended period of time. For example,the medication can be delivered to a patient over a period of timebetween 1 and 8 hours. The medication can also be delivered to a patientover a period of about 1, about 2, about 3, about 4, about 5, about 6,about 7, or about 8 hours. To accomplish an intravenous infusion, an IVgravity drip or an IV pump can be used. IV infusion is typically usedwhen a patient requires medications only at certain times and does notrequire additional intravenous fluids (e.g., water solutions which cancontain sodium, chloride, glucose, or any combination thereof) such asthose that restore electrolytes, blood sugar, and water loss.

C. Target Tissues

In some embodiments, the rhNaGlu of the invention is delivered to thecentral nervous system (CNS) of a subject. In some embodiments, therhNaGlu of the invention is delivered to one or more of target tissuesof brain, spinal cord, and/or peripheral organs. As used herein, theterm “target tissue” refers to any tissue that is affected by the NaGluassociated disease to be treated or any tissue in which the deficientNaGlu is normally expressed. In some embodiments, target tissues includethose tissues in which there is a detectable or abnormally high amountof enzyme substrate, for example stored in the cellular lysosomes of thetissue, in patients suffering from or susceptible to the NaGluassociated disease. In some embodiments, target tissues include thosetissues that display a disease-associated pathology, symptom, orfeature. In some embodiments, target tissues include those tissues inwhich the deficient NaGlu is normally expressed at an elevated level. Asused herein, a target tissue may be a brain target tissue, a spinal cordtarget tissue and/or a peripheral target tissue. Exemplary targettissues are described in detail below.

D. Brain Target Tissues

In general, the brain can be divided into different regions, layers andtissues. For example, meningeal tissue is a system of membranes whichenvelops the central nervous system, including the brain. The meningescontain three layers, including dura matter, arachnoid matter, and piamatter. In general, the primary function of the meninges and of thecerebrospinal fluid is to protect the central nervous system. In someembodiments, a therapeutic protein in accordance with the presentinvention is delivered to one or more layers of the meninges.

The brain has three primary subdivisions, including the cerebrum,cerebellum, and brain stem. The cerebral hemispheres, which are situatedabove most other brain structures and are covered with a cortical layer.Underneath the cerebrum lies the brainstem, which resembles a stalk onwhich the cerebrum is attached. At the rear of the brain, beneath thecerebrum and behind the brainstem, is the cerebellum.

The diencephalon, which is located near the midline of the brain andabove the mesencephalon, contains the thalamus, metathalamus,hypothalamus, epithalamus, prethalamus, and pretectum. Themesencephalon, also called the midbrain, contains the tectum,tegumentum, ventricular mesocoelia, and cerebral peduncels, the rednucleus, and the cranial nerve III nucleus. The mesencephalon isassociated with vision, hearing, motor control, sleep/wake, alertness,and temperature regulation.

Regions of tissues of the central nervous system, including the brain,can be characterized based on the depth of the tissues. For example, CNS(e.g., brain) tissues can be characterized as surface or shallowtissues, mid-depth tissues, and/or deep tissues.

According to the present invention, the rhNaGlu of the invention may bedelivered to any appropriate brain target tissue(s) associated with aparticular disease to be treated in a subject. In some embodiments, therhNaGlu of the invention is delivered to surface or shallow brain targettissue. In some embodiments, the rhNaGlu of the invention is deliveredto mid-depth brain target tissue. In some embodiments, the rhNaGlu ofthe invention is delivered to deep brain target tissue. In someembodiments, the rhNaGlu of the invention is delivered to a combinationof surface or shallow brain target tissue, mid-depth brain targettissue, and/or deep brain target tissue. In some embodiments, therhNaGlu of the invention is delivered to a deep brain tissue at least 4mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm or more below (or internal to)the external surface of the brain. Ranges and values intermediate to theabove recited ranges and values are also contemplated to be part of theinvention.

In some embodiments, the rhNaGlu of the invention is delivered to one ormore surface or shallow tissues of cerebrum. In some embodiments, thetargeted surface or shallow tissues of the cerebrum are located within 4mm from the surface of the cerebrum. In some embodiments, the targetedsurface or shallow tissues of the cerebrum are selected from pia matertissues, cerebral cortical ribbon tissues, hippocampus, Virchow Robinspace, blood vessels within the VR space, the hippocampus, portions ofthe hypothalamus on the inferior surface of the brain, the optic nervesand tracts, the olfactory bulb and projections, and combinationsthereof.

In some embodiments, the rhNaGlu of the invention is delivered to one ormore deep tissues of the cerebrum. In some embodiments, the targetedsurface or shallow tissues of the cerebrum are located at least 4 mm(e.g., 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm) below (or internal to)the surface of the cerebrum. In some embodiments, targeted deep tissuesof the cerebrum include the cerebral cortical ribbon. In someembodiments, targeted deep tissues of the cerebrum include one or moreof the diencephalon (e.g., the hypothalamus, thalamus, prethalamus orsubthalamus), metencephalon, lentiform nuclei, the basal ganglia,caudate, putamen, amygdala, globus pallidus, and combinations thereof.

In some embodiments, the rhNaGlu of the invention is delivered to one ormore tissues of the cerebellum. In certain embodiments, the targeted oneor more tissues of the cerebellum are selected from the group consistingof tissues of the molecular layer, tissues of the Purkinje cell layer,tissues of the Granular cell layer, cerebellar peduncles, andcombination thereof. In some embodiments, therapeutic agents (e.g.,enzymes) are delivered to one or more deep tissues of the cerebellumincluding, but not limited to, tissues of the Purkinje cell layer,tissues of the Granular cell layer, deep cerebellar white matter tissue(e.g., deep relative to the Granular cell layer), and deep cerebellarnuclei tissue.

In some embodiments, the rhNaGlu of the invention is delivered to one ormore tissues of the brainstem. In some embodiments, the targeted one ormore tissues of the brainstem include brain stem white matter tissueand/or brain stem nuclei tissue.

In some embodiments, the rhNaGlu of the invention is delivered tovarious brain tissues including, but not limited to, gray matter, whitematter, periventricular areas, pia-arachnoid, meninges, neocortex,cerebellum, deep tissues in cerebral cortex, molecular layer,caudate/putamen region, midbrain, deep regions of the pons or medulla,and combinations thereof.

In some embodiments, the rhNaGlu of the invention is delivered tovarious cells in the brain including, but not limited to, neurons, glialcells, perivascular cells and/or meningeal cells. In some embodiments, atherapeutic protein is delivered to oligodendrocytes of deep whitematter.

E. Spinal Cord Target Tissue

In general, regions or tissues of the spinal cord can be characterizedbased on the depth of the tissues. For example, spinal cord tissues canbe characterized as surface or shallow tissues, mid-depth tissues,and/or deep tissues.

In some embodiments, the rhNaGlu of the invention are delivered to oneor more surface or shallow tissues of the spinal cord. In someembodiments, a targeted surface or shallow tissue of the spinal cord islocated within 4 mm from the surface of the spinal cord. In someembodiments, a targeted surface or shallow tissue of the spinal cordcontains pia matter and/or the tracts of white matter.

In some embodiments, the rhNaGlu of the invention are delivered to oneor more deep tissues of the spinal cord. In some embodiments, a targeteddeep tissue of the spinal cord is located internal to 4 mm from thesurface of the spinal cord. In some embodiments, a targeted deep tissueof the spinal cord contains spinal cord grey matter and/or ependymalcells.

In some embodiments, replacement enzymes (e.g., a NaGlu fusion protein)are delivered to neurons of the spinal cord.

F. Peripheral Target Tissues

As used herein, peripheral organs or tissues refer to any organs ortissues that are not part of the central nervous system (CNS).Peripheral target tissues may include, but are not limited to, bloodsystem, liver, kidney, heart, endothelium, bone marrow and bone marrowderived cells, spleen, lung, lymph node, bone, cartilage, ovary andtestis. In some embodiments, the rhNaGlu of the invention is deliveredto one or more of the peripheral target tissues.

G. Biodistribution and Bioavailability

In various embodiments, once delivered to the target tissue, the rhNaGluof the invention is localized intracellularly. For example, the rhNaGluof the invention may be localized to exons, axons, lysosomes,mitochondria or vacuoles of a target cell (e.g., neurons such asPurkinje cells). For example, in some embodiments the rhNaGlu of theinvention demonstrates translocation dynamics such that the rhNaGlumoves within the perivascular space (e.g., by pulsation-assistedconvective mechanisms). In addition, active axonal transport mechanismsrelating to the association of the administered protein or enzyme withneurofilaments may also contribute to or otherwise facilitate thedistribution of the rhNaGlu proteins of the invention into the deepertissues of the central nervous system.

In some embodiments, the rhNaGlu of the invention delivered according tothe present invention may achieve therapeutically or clinicallyeffective levels or activities in various targets tissues describedherein. As used herein, a therapeutically or clinically effective levelor activity is a level or activity sufficient to confer a therapeuticeffect in a target tissue. The therapeutic effect may be objective(i.e., measurable by some test or marker) or subjective (i.e., subjectgives an indication of or feels an effect). For example, atherapeutically or clinically effective level or activity may be anenzymatic level or activity that is sufficient to ameliorate symptomsassociated with the disease in the target tissue (e.g., GAG storage).

In some embodiments, the rhNaGlu of the invention delivered according tothe present invention may achieve an enzymatic level or activity that isat least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the normal level or activity of thecorresponding NaGlu enzyme in the target tissue. In some embodiments,the rhNaGlu of the invention delivered according to the presentinvention may achieve an enzymatic level or activity that is increasedby at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold or 10-fold as compared to a control (e.g., endogenouslevels or activities without the treatment). In some embodiments, therhNaGlu delivered according to the present invention may achieve anincreased enzymatic level or activity at least approximately 10nmol/hr/mg, 20 nmol/hr/mg, 40 nmol/hr/mg, 50 nmol/hr/mg, 60 nmol/hr/mg,70 nmol/hr/mg, 80 nmol/hr/mg, 90 nmol/hr/mg, 100 nmol/hr/mg, 150nmol/hr/mg, 200 nmol/hr/mg, 250 nmol/hr/mg, 300 nmol/hr/mg, 350nmol/hr/mg, 400 nmol/hr/mg, 450 nmol/hr/mg, 500 nmol/hr/mg, 550nmol/hr/mg or 600 nmol/hr/mg in a target tissue. Ranges and valuesintermediate to the above recited ranges and values are alsocontemplated to be part of the invention.

In some embodiments, inventive methods according to the presentinvention are particularly useful for targeting the lumbar region. Insome embodiments, the rhNaGlu delivered according to the presentinvention may achieve an increased enzymatic level or activity in thelumbar region of at least approximately 500 nmol/hr/mg, 600 nmol/hr/mg,700 nmol/hr/mg, 800 nmol/hr/mg, 900 nmol/hr/mg, 1000 nmol/hr/mg, 1500nmol/hr/mg, 2000 nmol/hr/mg, 3000 nmol/hr/mg, 4000 nmol/hr/mg, 5000nmol/hr/mg, 6000 nmol/hr/mg, 7000 nmol/hr/mg, 8000 nmol/hr/mg, 9000nmol/hr/mg, or 10,000 nmol/hr/mg. Ranges and values intermediate to theabove recited ranges and values are also contemplated to be part of theinvention.

In general, therapeutic agents (e.g., the rhNaGlu) delivered accordingto the present invention have sufficiently long half time in CSF andtarget tissues of the brain, spinal cord, and peripheral organs. In someembodiments, the rhNaGlu delivered according to the present inventionmay have a half-life of at least approximately 30 minutes, 45 minutes,60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7hours, 8 hours, 9 hours, 10 hours, 12 hours, 16 hours, 18 hours, 20hours, 25 hours, 30 hours, 35 hours, 40 hours, up to 3 days, up to 7days, up to 14 days, up to 21 days or up to a month. In someembodiments, In some embodiments, the rhNaGlu delivered according to thepresent invention may retain detectable level or activity in CSF orbloodstream after 12 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90hours, 96 hours, 102 hours, or a week following administration.Detectable level or activity may be determined using various methodsknown in the art. Ranges and values intermediate to the above recitedranges and values are also contemplated to be part of the invention. Incertain embodiments, the rhNaGlu delivered according to the presentinvention achieves a concentration of at least 30 μg/mL in the CNStissues and cells of the subject following administration (e.g., oneweek, 3 days, 48 hours, 36 hours, 24 hours, 18 hours, 12 hours, 8 hours,6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, or less,following administration of the pharmaceutical composition to thesubject). In certain embodiments, the rhNaGlu delivered according to thepresent invention achieves a concentration of at least 2 μg/mL, at least15 μg/mL, at least 1 μg/mL, at least 7 μg/mL, at least 5 μg/mL, at least2 μg/mL, at least 1 μg/mL or at least 0.5 μg/mL in the targeted tissuesor cells of the subject (e.g., brain tissues or neurons) followingadministration to such subject (e.g., one week, 3 days, 48 hours, 36hours, 24 hours, 18 hours, 12 hours, 8 hours, 6 hours, 4 hours, 3 hours,2 hours, 1 hour, 30 minutes, or less following administration of suchpharmaceutical compositions to the subject). Ranges and valuesintermediate to the above recited ranges and values are alsocontemplated to be part of the invention.

H. Treatment of Sanfilippo Syndrome

Sanfilippo syndrome, or mucopolysaccharidosis III (MPS III), is a raregenetic disorder characterized by the deficiency of enzymes involved inthe degradation of glycosaminoglycans (GAG). In the absence of enzyme,partially degraded GAG molecules cannot be cleared from the body andaccumulate in lysosomes of various tissues, resulting in progressivewidespread somatic dysfunction (Neufeld and Muenzer, 2001).

Four distinct forms of MPS III, designated MPS IIIA, B, C, and D, havebeen identified. Each represents a deficiency in one of four enzymesinvolved in the degradation of the GAG heparan sulfate. All formsinclude varying degrees of the same clinical symptoms, including coarsefacial features, hepatosplenomegaly, corneal clouding and skeletaldeformities. Most notably, however, is the severe and progressive lossof cognitive ability, which is tied not only to the accumulation ofheparan sulfate in neurons, but also the subsequent elevation of thegangliosides GM2, GM3 and GD2 caused by primary GAG accumulation(Walkley 1998).

Mucopolysaccharidosis type 111B (MPS IIIB; Sanfilippo syndrome B) is anautosomal recessive disorder that is characterized by a deficiency ofthe enzyme alpha-N-acetyl-glucosaminidase (NaGlu). In the absence ofthis enzyme, GAG heparan sulfate accumulates in lysosomes of neurons andglial cells, with lesser accumulation outside the brain.

A defining clinical feature of this disorder is central nervous system(CNS) degeneration, which results in loss of, or failure to attain,major developmental milestones. The progressive cognitive declineculminates in dementia and premature mortality. The disease typicallymanifests itself in young children, and the lifespan of an affectedindividual generally does not extend beyond late teens to earlytwenties.

Compositions and methods of the present invention may be used toeffectively treat individuals suffering from or susceptible toSanfilippo syndrome B. The terms, “treat” or “treatment,” as usedherein, refers to amelioration of one or more symptoms associated withthe disease, prevention or delay of the onset of one or more symptoms ofthe disease, and/or lessening of the severity or frequency of one ormore symptoms of the disease.

In some embodiments, treatment refers to partial or completealleviation, amelioration, relief, inhibition, delaying onset, reducingseverity and/or incidence of neurological impairment in a Sanfilipposyndrome B patient. As used herein, the term “neurological impairment”includes various symptoms associated with impairment of the centralnervous system (e.g., the brain and spinal cord). Symptoms ofneurological impairment may include, for example, developmental delay,progressive cognitive impairment, hearing loss, impaired speechdevelopment, deficits in motor skills, hyperactivity, aggressivenessand/or sleep disturbances, among others.

Thus, in some embodiments, treatment refers to decreased lysosomalstorage (e.g., of GAG) in various tissues. In some embodiments,treatment refers to decreased lysosomal storage in brain target tissues,spinal cord neurons, and/or peripheral target tissues. In certainembodiments, lysosomal storage is decreased by about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 100% or more as compared to a control. In some embodiments,lysosomal storage is decreased by at least 1-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold as compared toa control. In some embodiments, lysosomal storage is determined byLAMP-1 staining. Ranges and values intermediate to the above recitedranges and values are also contemplated to be part of the invention.

In some embodiments, treatment refers to reduced vacuolization inneurons

(e.g., neurons containing Purkinje cells). In certain embodiments,vacuolization in neurons is decreased by about 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100% or more as compared to a control. In some embodiments,vacuolization is decreased by at least 1-fold, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold as compared to acontrol. Ranges and values intermediate to the above recited ranges andvalues are also contemplated to be part of the invention.

In some embodiments, treatment refers to increased NaGlu enzyme activityin various tissues. In some embodiments, treatment refers to increasedNaGlu enzyme activity in brain target tissues, spinal cord neuronsand/or peripheral target tissues. In some embodiments, NaGlu enzymeactivity is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%,400%, 500%, 600%, 700%, 800%, 900% 1000% or more as compared to acontrol. In some embodiments, NaGlu enzyme activity is increased by atleast 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold or 10-fold as compared to a control. In some embodiments,increased NaGlu enzymatic activity is at least approximately 10nmol/hr/mg, 20 nmol/hr/mg, 40 nmol/hr/mg, 50 nmol/hr/mg, 60 nmol/hr/mg,70 nmol/hr/mg, 80 nmol/hr/mg, 90 nmol/hr/mg, 100 nmol/hr/mg, 150nmol/hr/mg, 200 nmol/hr/mg, 250 nmol/hr/mg, 300 nmol/hr/mg, 350nmol/hr/mg, 400 nmol/hr/mg, 450 nmol/hr/mg, 500 nmol/hr/mg, 550nmol/hr/mg, 600 nmol/hr/mg or more. In some embodiments, NaGlu enzymaticactivity is increased in the lumbar region. In some embodiments,increased NaGlu enzymatic activity in the lumbar region is at leastapproximately 2000 nmol/hr/mg, 3000 nmol/hr/mg, 4000 nmol/hr/mg, 5000nmol/hr/mg, 6000 nmol/hr/mg, 7000 nmol/hr/mg, 8000 nmol/hr/mg, 9000nmol/hr/mg, 10,000 nmol/hr/mg, or more. Ranges and values intermediateto the above recited ranges and values are also contemplated to be partof the invention.

In certain embodiments, treatment according to the present inventionresults in a reduction (e.g., about a 5%, 10%, 15%, 20%, 25%, 30%, 40%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 97.5%, 99% or morereduction) or a complete elimination of the presence, or alternativelythe accumulation, of one or more pathological or biological markerswhich are associated with the NaGlu associated disease. Such reductionor elimination may be particularly evident in the cells and tissues ofthe CNS (e.g., neurons and oligodendrocytes). For example, in someembodiments, upon administration to a subject the pharmaceuticalcompositions of the present invention demonstrate or achieve a reductionin the accumulation of the biomarker lysosomal associated membraneprotein 1 (LAMP1) in the CNS cells and tissues of the subject (e.g., inthe cerebral cortex, cerebellum, caudate nucleus and putamen, whitematter and/or thalamus). LAMP1 is a glycoprotein highly expressed inlysosomal membranes and its presence is elevated many patients with alysosomal storage disorder (Meikle et al., Clin. Chem. (1997)43:1325-1335). The presence or absence of LAMP 1 in patients (e.g., asdetermined by LAMP staining) with a lysosomal storage disease thereforemay provide a useful indicator of lysosomal activity and a marker forboth the diagnosis and monitoring of lysosomal storage diseases.

Accordingly, some embodiments of the present invention relate to methodsof reducing or otherwise eliminating the presence or accumulation of oneor more pathological or biological markers associated with the NaGluassociated disease. Similarly, some embodiments of the invention relateto methods of increasing the degradation (or the rate of degradation) ofone or more pathological or biological markers (e.g., LAMP1) associatedwith lysosomal storage diseases.

In some embodiments, treatment refers to decreased progression of lossof cognitive ability. In certain embodiments, progression of loss ofcognitive ability is decreased by about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% ormore as compared to a control. In some embodiments, treatment refers todecreased developmental delay. In certain embodiments, developmentaldelay is decreased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more ascompared to a control. Ranges and values intermediate to the aboverecited ranges and values are also contemplated to be part of theinvention.

In some embodiments, treatment refers to increased survival (e.g.,survival time). For example, treatment can result in an increased lifeexpectancy of a patient. In some embodiments, treatment according to thepresent invention results in an increased life expectancy of a patientby more than about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about95%, about 100%, about 105%, about 110%, about 115%, about 120%, about125%, about 130%, about 135%, about 140%, about 145%, about 150%, about155%, about 160%, about 165%, about 170%, about 175%, about 180%, about185%, about 190%, about 195%, about 200% or more, as compared to theaverage life expectancy of one or more control individuals with similardisease without treatment. In some embodiments, treatment according tothe present invention results in an increased life expectancy of apatient by more than about 6 month, about 7 months, about 8 months,about 9 months, about 10 months, about 11 months, about 12 months, about2 years, about 3 years, about 4 years, about 5 years, about 6 years,about 7 years, about 8 years, about 9 years, about 10 years or more, ascompared to the average life expectancy of one or more controlindividuals with similar disease without treatment. In some embodiments,treatment according to the present invention results in long termsurvival of a patient. As used herein, the term “long term survival”refers to a survival time or life expectancy longer than about 40 years,45 years, 50 years, 55 years, 60 years, or longer. Ranges and valuesintermediate to the above recited ranges and values are alsocontemplated to be part of the invention.

The terms, “improve,” “increase” or “reduce,” as used herein, indicatevalues that are relative to a control. In some embodiments, a suitablecontrol is a baseline measurement, such as a measurement in the sameindividual prior to initiation of the treatment described herein, or ameasurement in a control individual (or multiple control individuals) inthe absence of the treatment described herein. A “control individual” isan individual afflicted with Sanfilippo syndrome B, who is about thesame age and/or gender as the individual being treated (to ensure thatthe stages of the disease in the treated individual and the controlindividual(s) are comparable).

The individual (also referred to as “patient” or “subject”) beingtreated is an individual (fetus, infant, child, adolescent, or adulthuman) having Sanfilippo syndrome B or having the potential to developSanfilippo syndrome B. The individual can have residual endogenous NaGluexpression and/or activity, or no measurable activity. For example, theindividual having Sanfilippo Syndrome B may have NaGlu expression levelsthat are less than about 30-50%, less than about 25-30%, less than about20-25%, less than about 15-20%, less than about 10-15%, less than about5-10%, less than about 0.1-5% of normal NaGlu expression levels. Rangesand values intermediate to the above recited ranges and values are alsocontemplated to be part of the invention.

In some embodiments, the individual is an individual who has beenrecently diagnosed with the disease. Typically, early treatment(treatment commencing as soon as possible after diagnosis) is importantto minimize the effects of the disease and to maximize the benefits oftreatment.

I. Combination Therapies

Recombinant human NaGlu proteins, for instance a recombinant human NaGluprotein containing a sufficient amount of oligosaccharides (e.g.,mannose and phosphorylated mannose (i.e., M6P)), can be used alone or incombination to treat NaGlu associated diseases (e.g., SanfilippoSyndrome B). It should be understood that the recombinant human NaGluproteins of the invention can be used alone or in combination with anadditional procedure, e.g., surgical procedure, or agent, e.g.,therapeutic agent, the additional procedure or agent being selected bythe skilled artisan for its intended purpose. For instance, theadditional procedure or agent can be a therapeutic procedure or agentart-recognized as being useful to treat the disease or condition beingtreated by the recombinant human NaGlu protein of the present invention.The additional procedure or agent also can be an agent that imparts abeneficial attribute to the therapeutic composition, e.g., an agentwhich affects the viscosity of the composition.

It should also be understood that the combinations which are includedwithin this invention are those combinations useful for their intendedpurpose. The agents and procedures set forth below are for illustrativepurposes and not intended to be limiting to the present invention. Thecombinations, which are part of this invention, can be the recombinanthuman NaGlu proteins of the present invention and at least oneadditional agent or procedure selected from the lists below. Thecombination can also include more than one additional agent orprocedure, e.g., two or three additional agents if the combination issuch that the formed composition can perform its intended function.

The combination therapy can include surgical procedures, gene therapy,or enzyme-replacement therapy. Additionally, the recombinant human NaGluprotein can be coformulated with one or more additional therapeuticagents, e.g., other recombinant proteins or antibodies or drugs capableof preventing or reducing the accumulation of undegraded substrates(e.g., substrate reduction therapy).

In one or more embodiments, the combination therapy can includeco-administration with immunosuppresants, as discussed in further detailbelow. Immunosuppresants such as, but not limited to, antihistamines,corticosteroids, sirolimus, voclosporin, ciclosporin, methotrexate, IL-2receptor directed antibodies, T-cell receptor directed antibodies,TNF-alpha directed antibodies or fusion proteins (e.g., infliximab,etanercept, or adalimumab), CTLA-4-Ig (e.g., abatacept), anti-OX-40antibodies can also be administered before, during, or afteradministration of a recombinant human protein, such as a recombinanthuman NaGlu protein, for example, if an anaphylactic reaction or adverseimmune response is expected or experienced by a patient.

J. Immunogenicity

The pharmaceutical compositions of the present invention arecharacterized by their tolerability. As used herein, the terms“tolerable” and “tolerability” refer to the ability of thepharmaceutical compositions of the present invention to not elicit anadverse reaction in the subject to whom such composition isadministered, or alternatively not to elicit a serious adverse reactionin the subject to whom such composition is administered. In someembodiments, the pharmaceutical compositions of the present inventionare well tolerated by the subject to whom such compositions isadministered.

Generally, administration of a rhNaGlu protein according to the presentinvention does not result in severe adverse effects in the subject. Asused herein, severe adverse effects induce, but are not limited to,substantial immune response, toxicity, or death. As used herein, theterm “substantial immune response” refers to severe or serious immuneresponses, such as adaptive T-cell immune responses.

Thus, in many embodiments, inventive methods according to the presentinvention do not involve concurrent immunosuppressant therapy (i.e., anyimmunosuppressant therapy used as pre-treatment/pre-conditioning or inparallel to the method). In some embodiments, inventive methodsaccording to the present invention do not involve an immune toleranceinduction in the subject being treated. In some embodiments, inventivemethods according to the present invention do not involve apre-treatment or preconditioning of the subject using T-cellimmunosuppressive agent.

However, in some embodiments, a subject mounts an immune response afterbeing administered the rhNaGlu of the invention. Thus, in someembodiments, it may be useful to render the subject receiving therhNaGlu of the invention tolerant to the enzyme replacement therapyImmune tolerance may be induced using various methods known in the art.For example, an initial 30-60 day regimen of a T-cell immunosuppressiveagent such as cyclosporin A (CsA) and an antiproliferative agent, suchas, azathioprine (Aza), combined with weekly intrathecal infusions oflow doses of a desired replacement enzyme may be used.

Any immunosuppressant agent known to the skilled artisan may be employedtogether with a combination therapy of the invention. Suchimmunosuppressant agents include but are not limited to cyclosporine,FK506, rapamycin, CTLA4-Ig, and anti-TNF agents such as etanercept (seee.g., Moder, 2000, Ann. Allergy Asthma Immunol. 84, 280-284; Nevins,2000, Curr. Opin. Pediatr. 12, 146-150; Kurlberg et al., 2000, Scand. J.Immunol. 51, 224-230; Ideguchi et al., 2000, Neuroscience 95, 217-226;Potter et al., 1999, Ann N.Y. Acad. Sci. 875, 159-174; Slavik et al.,1999, Immunol. Res. 19, 1-24; Gaziev et al., 1999, Bone MarrowTransplant. 25, 689-696; Henry, 1999, Clin. Transplant. 13, 209-220;Gummert et al., 1999, J. Am. Soc. Nephrol. 10, 1366-1380; Qi et al.,2000, Transplantation 69, 1275-1283). The anti-IL2 receptor (a-subunit)antibody daclizumab (e.g., Zenapax™), which has been demonstratedeffective in transplant patients, can also be used as animmunosuppressant agent (see e.g., Wiseman et al., 1999, Drugs 58,1029-1042; Beniaminovitz et al., 2000, N. Engl J. Med. 342, 613-619;Ponticelli et al., 1999, Drugs R. D. 1, 55-60; Berard et al., 1999,Pharmacotherapy 19, 1 127-1 137; Eckhoff et al., 2000, Transplantation69, 1867-1872; Ekberg et al., 2000, Transpl. Int. 13, 151-159).Additional immunosuppressant agents include but are not limited toanti-CD2 (Branco et al., 1999, Transplantation 68, 1588-1596; Przepiorkaet al., 1998, Blood 92, 4066-4071), anti-CD4 (Marinova-Mutafchieva etal., 2000, Arthritis Rheum. 43, 638-644; Fishwild et al., 1999, Clin.Immunol. 92, 138-152), and anti-CD40 ligand (Hong et al., 2000, SeminNephrol. 20, 108-125; Chirmule et al., 2000, J. Virol. 74, 3345-3352;Ito et al., 2000, J. Immunol. 164, 1230-1235).

In other embodiments, the invention includes methods comprisingco-administration of the NaGlu proteins of the present invention withagents which decrease or suppress an immune response to the NaGluprotein, e.g., immunosuppresants Immunosuppresants such as, but notlimited to, antihistamines, corticosteroids, sirolimus, voclosporin,ciclosporin, methotrexate, IL-2 receptor directed antibodies, T-cellreceptor directed antibodies, TNF-alpha directed antibodies or fusionproteins (e.g., infliximab, etanercept, or adalimumab), CTLA-4-Ig (e.g.,abatacept), anti-OX-40 antibodies can also be administered before,during, or after administration of a recombinant human protein, such asa recombinant human NaGlu protein, for example, if an anaphylacticreaction or adverse immune response is expected or experienced by apatient.

In one embodiment, the invention provides for a pretreatment procedureto minimize or prevent any potential anaphylactic reactions that can beincurred by administration of the recombinant protein in accordance withthe invention. In one embodiment, to prevent a potential anaphylacticreaction, an H-1 receptor antagonist, also known as an antihistamine(e.g., diphenhydramine) is administered to the patient.

In one embodiment, the H-1 receptor antagonist is administered in a doseof about 1 mg to about 10 mg per kilogram of body weight. For example,an antihistamine can be administered in a dose of about 5 mg perkilogram. In one embodiment, the antihistamine is administered in a doseof between about 0.1 mg and about 10 mg per kilogram of body weight. Inone embodiment, the antihistamine is administered in a dose betweenabout 1 mg and about 5 mg per kilogram of body weight. For example thedose can be 1 mg, 2 mg, 3 mg, 4 mg, or 5 mg per kilogram of body weight.The antihistamine can be administered by any useful method. In oneembodiment, the antihistamine is administered intravenously. In anotherembodiment, the antihistamine is administered in pharmaceuticallyacceptable capsules.

Administration of the antihistamine can be prior to the administrationof the recombinant NaGlu in accordance with the invention. In oneembodiment, the H-1 receptor antagonist is administered about 10 toabout 90 minutes, for example, about 30 to about 60 minutes prior to theadministration of recombinant NaGlu. The H-1 receptor antagonist can beadministered using an ambulatory system connected to a vascular accessport. In one embodiment, the antihistamine is administered about 90minutes prior to the administration of recombinant NaGlu. In oneembodiment, the antihistamine is administered between about 10 and about60 minutes prior to the administration of recombinant NaGlu. In anotherembodiment, the antihistamine is administered between about 20 and about40 minutes prior to administering recombinant NaGlu. For example, theantihistamine can be administered 20, 25, 30, 35, or 40 minutes prior tothe administration of recombinant NaGlu.

In one embodiment, the antihistamine administered is diphenhydramine.Any useful antihistamine can be used. Such antihistamines include,without limitation, clemastine, doxylamine, loratidine, desloratidine,fexofenadine, pheniramine, cetirizine, ebastine, promethazine,chlorpheniramine, levocetirizine, olopatadine, quetiapine, meclizine,dimenhydrinate, embramine, dimethidene, and dexchloropheniramine

In another embodiment, with reference to intravenous infusion, thepotential for anaphylactic reactions can be reduced by administering theinfusions using a ramp-up protocol. In this context, a ramp-up protocolrefers to slowly increasing the rate of the infusion over the course ofthe infusion in order to desensitize the patient to the infusion of themedication.

K. Administration

The methods of the present invention contemplate single as well asmultiple administrations of a therapeutically effective amount of therhNaGlu of the invention described herein. The rhNaGlu of the inventioncan be administered at regular intervals, depending on the nature,severity and extent of the subject's condition. In some embodiments, atherapeutically effective amount of the rhNaGlu protein of the presentinvention may be administered intravenously or intrathecallyperiodically at regular intervals (e.g., once every year, once every sixmonths, once every five months, once every three months, bimonthly (onceevery two months), monthly (once every month), biweekly (once every twoweeks) or weekly).

In some embodiments, intrathecal administration may be used inconjunction with other routes of administration (e.g., intravenous,subcutaneously, intramuscularly, parenterally, trans dermally, ortransmucosally (e.g., orally or nasally)). In some embodiments, thoseother routes of administration (e.g., intravenous administration) may beperformed no more frequent than biweekly, monthly, once every twomonths, once every three months, once every four months, once every fivemonths, once every six months, annually administration.

As used herein, the term “therapeutically effective amount” is largelydetermined based on the total amount of the therapeutic agent containedin the pharmaceutical compositions of the present invention. Generally,a therapeutically effective amount is sufficient to achieve a meaningfulbenefit to the subject (e.g., treating, modulating, curing, preventingand/or ameliorating the underlying disease or condition). For example, atherapeutically effective amount may be an amount sufficient to achievea desired therapeutic and/or prophylactic effect, such as an amountsufficient to modulate lysosomal enzyme receptors or their activity tothereby treat such lysosomal storage disease or the symptoms thereof(e.g., a reduction in or elimination of the presence or incidence of“zebra bodies” or cellular vacuolization following the administration ofthe compositions of the present invention to a subject). Generally, theamount of a therapeutic agent (e.g., the rhNaGlu of the invention)administered to a subject in need thereof will depend upon thecharacteristics of the subject. Such characteristics include thecondition, disease severity, general health, age, sex and body weight ofthe subject. One of ordinary skill in the art will be readily able todetermine appropriate dosages depending on these and other relatedfactors. In addition, both objective and subjective assays mayoptionally be employed to identify optimal dosage ranges.

A therapeutically effective amount is commonly administered in a dosingregimen that may comprise multiple unit doses. For any particulartherapeutic protein, a therapeutically effective amount (and/or anappropriate unit dose within an effective dosing regimen) may vary, forexample, depending on route of administration, on combination with otherpharmaceutical agents. Also, the specific therapeutically effectiveamount (and/or unit dose) for any particular patient may depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific pharmaceutical agentemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and/or rate of excretion or metabolism of thespecific fusion protein employed; the duration of the treatment; andlike factors as is well known in the medical arts.

In some embodiments, the therapeutically effective dose ranges fromabout 0.005 mg/kg body weight to 500 mg/kg body weight, e.g., from about0.005 mg/kg body weight to 400 mg/kg body weight, from about 0.005 mg/kgbody weight to 300 mg/kg body weight, from about 0.005 mg/kg body weightto 200 mg/kg body weight, from about 0.005 mg/kg body weight to 100mg/kg body weight, from about 0.005 mg/kg body weight to 90 mg/kg bodyweight, from about 0.005 mg/kg body weight to 80 mg/kg body weight, fromabout 0.005 mg/kg body weight to 70 mg/kg body weight, from about 0.005mg/kg body weight to 60 mg/kg body weight, from about 0.005 mg/kg bodyweight to 50 mg/kg body weight, from about 0.005 mg/kg body weight to 40mg/kg body weight, from about 0.005 mg/kg body weight to 30 mg/kg bodyweight, from about 0.005 mg/kg body weight to 25 mg/kg body weight, fromabout 0.005 mg/kg body weight to 20 mg/kg body weight, from about 0.005mg/kg body weight to 15 mg/kg body weight, from about 0.005 mg/kg bodyweight to 10 mg/kg bra body in weight. Ranges and values intermediate tothe above recited ranges and values (e.g., 10-50 mg/kg, 1-5 mg/kg, 2-8mg/kg, 5-10 mg/kg, 0.1-10 mg/kg, 0.3-30 mg/kg, 0.3-50 mg/kg, 0.5-10mg/kg, 5-30 mg/kg, or 6-27 mg/kg) are also contemplated to be part ofthe invention.

In some embodiments, the therapeutically effective dose is greater thanor at least about 0.1 mg/kg body weight, greater than or at least about0.2 mg/kg body weight, greater than or at least about 0.3 mg/kg bodyweight, greater than or at least about 0.4 mg/kg body weight, greaterthan or at least about 0.5 mg/kg body weight, greater than or at leastabout 1.0 mg/kg body weight, greater than or at least about 3 mg/kg bodyweight, greater than or at least about 5 mg/kg body weight, greater thanor at least about 6 mg/kg body weight, greater than or at least about 7mg/kg body weight greater than or at least about 10 mg/kg body weight,greater than or at least about 15 mg/kg body weight, greater than or atleast about 20 mg/kg body weight, greater than or at least about 30mg/kg body weight, greater than or at least about 40 mg/kg body weight,greater than or at least about 50 mg/kg body weight, greater than or atleast about 60 mg/kg body weight, greater than or at least about 70mg/kg body weight, greater than about or at least 80 mg/kg body weight,greater than or at least about 90 mg/kg body weight, greater than or atleast about 100 mg/kg body weight. Ranges and values intermediate to theabove recited ranges and values are also contemplated to be part of theinvention.

In some embodiments, the therapeutically effective dose may also bedefined by mg/kg brain weight. As one skilled in the art wouldappreciate, the brain weights and body weights can be correlated (see,e.g., Dekaban AS. “Changes in brain weights during the span of humanlife: relation of brain weights to body heights and body weights,” AnnNeurol 1978; 4:345-56).

In some embodiments, the therapeutically effective dose may also bedefined by mg/15 cc of CSF. As one skilled in the art would appreciate,therapeutically effective doses based on brain weights and body weightscan be converted to mg/15 cc of CSF. For example, the volume of CSF inadult humans is approximately 150 mL (Johanson C E, et al. “Multiplicityof cerebrospinal fluid functions: New challenges in health and disease,”Cerebrospinal Fluid Res. 2008 May 14; 5: 10). Therefore, single doseinjections of 0.1 mg to 50 mg protein to adults would be approximately0.01 mg/15 cc of CSF (0.1 mg) to 5.0 mg/15 cc of CSF (50 mg) doses inadults.

It is to be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the enzyme replacement therapy andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed invention.

VIII. Kits

The present invention further provides kits or other articles ofmanufacture which contain the recombinant human NaGlu of the presentinvention and provide instructions for its reconstitution (iflyophilized) and/or use. Kits or other articles of manufacture mayinclude a container, a catheter and any other articles, devices orequipment useful in intrathecal administration and associated surgery.Suitable containers include, for example, bottles, vials, syringes(e.g., pre-filled syringes), ampules, cartridges, reservoirs, orlyo-jects. The container may be formed from a variety of materials suchas glass or plastic. In some embodiments, a container is a pre-filledsyringe. Suitable pre-filled syringes include, but are not limited to,borosilicate glass syringes with baked silicone coating, borosilicateglass syringes with sprayed silicone, or plastic resin syringes withoutsilicone.

Typically, a label on, or associated with, the container may indicatedirections for use and/or reconstitution. For example, the label mayindicate that the formulation is reconstituted to protein concentrationsas described above. The label may further indicate that the formulationis useful or intended for, for example, intravenous or intrathecaladministration. In some embodiments, a container may contain a singledose of a stable formulation containing a replacement enzyme (e.g., arecombinant NaGlu protein). In various embodiments, a single dose of thestable formulation is present in a volume of less than about 15 mL, 10mL, 5.0 mL, 4.0 mL, 3.5 mL, 3.0 mL, 2.5 mL, 2.0 mL, 1.5 mL, 1.0 mL, or0.5 mL. Alternatively, a container holding the formulation may be amulti-use vial, which allows for repeat administrations (e.g., from 2-6administrations) of the formulation. Kits or other articles ofmanufacture may further include a second container comprising a suitablediluent (e.g., BWFI, saline, buffered saline). Upon mixing of thediluent and the formulation, the final protein concentration in thereconstituted formulation will generally be at least 1 mg/mL (e.g., atleast 5 mg/mL, at least 10 mg/mL, at least 25 mg/mL, at least 50 mg/mL,at least 75 mg/mL, at least 100 mg/mL).

Kits or other articles of manufacture may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, catheters, syringes, andpackage inserts with instructions for use. Ranges and valuesintermediate to the above recited ranges and values are alsocontemplated to be part of the invention.

EXAMPLES

The following specific examples are intended to illustrate the inventionand should not be construed as limiting the scope of the claims. Thecontents of all figures and all references, patents and published patentapplications cited throughout this application, as well as the Figures,are expressly incorporated herein by reference in their entirety.

Example 1 Purification of rhNaGlu

rhNaGlu protein was purified by using methods known in the art. Eggwhite (EW) containing rhNaGlu was solubilized at pH 6 overnight andclarified through centrifugation and/or depth filtration. The EW wasadjusted with 1 M NaOAc buffer (pH 4) to pH 6. For the depth filtrationprocess, T2600 filter (Pall™, 40 um) was used as a 1^(st) filtration andthen PDF1 (Pall™, K200P, 15 um+EKS, 0.22 um) as a 2^(nd) filtrationstep. The filters are single-use membrane with an optimized capacity 60L EW/m² for each filter. The hold volume of membrane is 2 L/m² for T2600and 4-5 L/m² for PDF1. In the process, the hold volume was discardedbefore the filtered EW collected. The buffer (20 mM Phosphate/137 mMNaCl, pH 6) equivalent to the membrane hold volume was used to chase EWleft on the filters.

A phenyl-HIC (hydrophobic interaction chromatography) column was appliedas a capture step. Since most of egg white proteins are hydrophilic, 99%of egg white proteins passed through the HIC column into flow through.rhNaGlu has a higher hydrophobicity binding to phenyl-HIC.

Egg white containing rhNaGlu was loaded onto the column with a ratio of30:1. After completion of loading, the column was washed with theequilibration buffer, 5 mM phosphate buffer, pH 6, and 5 mM Tris buffer,pH 7.2. rhNaGlu was eluted with 30% propylene glycol, pH 7.2. After thecompletion of loading, the column was washed with equilibration bufferand 5 mM phosphate buffer (pH 6). rhNaGlu was eluted with 30% propyleneglycol with 5 mM Tris buffer (pH 7.2). The column binding capacity isapproximately 4.5 mg/mL. The purity of rhNaGlu through the phenyl-HICcolumn can be reached to >95% (950 time increase). The recovery isapproximately 80% with 30% of propylene glycol elution.

The eluted rhNaGlu fraction was adjusted to pH 5 with 1 M acetic acidand then loaded onto a GigaCap S column (EW: column size=10:1). Thecolumn was equilibrated with 50 mM NaOAc buffer (pH 5). After completionof loading, the column was washed with the equilibration buffer. TherhNaGlu was eluted with 50 mM NaOAc/60 mM NaCl (pH 5).

The protein characterization was performed using purified rhNaGlu. Themolecular weight of rhNaGlu (−90 kDa) purified from egg white wasanalyzed on SDS-PAGE (FIG. 6). The average expression level of rhNaGluin egg white is shown in FIG. 7. The characteristics of rhNaGlu producedfrom the transgenic avian are summarized in Table 2.

TABLE 2 rhNaGlu (Gallus) Apparent Molecular Weight ~90 kDa pI 6.1-6.9 pHStability pH 5-8 Stability in Egg White >50 days

Example 2 Stability of rhNaGlu in Egg White

A single egg was cracked 7 days post-lay and analyzed for activity.Contents were divided in half and each half was subject to standard eggwhite clarification. Both untreated and clarified egg whites werealiquoted and stored at 4° C. and −20° C. for enzyme activity stability.rhNaGlu in egg white showed stable enzyme activity at least up to 50days.

Freeze/thaw cycle stability was assessed. The purified rhNaGlu wasfrozen in liquid nitrogen for 10 seconds and thawed at 37° C. for 2 min.The enzyme activity showed no change for 10 cycles.

The purified rhNaGlu was dialyzed into different pH buffers to measurethe stability of pure enzyme. The results showed that pure rhNaGlu wasstable between pH 5-8 for 12 days.

Example 3 Oligosaccharide Profiling

Mannose-6-phosphate (M6P) is a terminal monosaccharide of N-linkedoligosaccharides that is an important part of the tertiary structure ofglycoprotein and, when incorporated in the glycoprotein's finaloligosaccharide, is recognized by and bound to the M6P receptors presenton the cell surface, subsequently allowing internalization into thelysosomes. Thus, M6P is an effective epitope for the targeting ofglycoproteins to the lysosomes.

Analysis of protein glycosylation is an important part of glycoproteincharacterization. Oligosaccharides can be linked to a protein through aserine or a threonine as O-lined glycans or through an asparagine asN-linked glycans.

To analyze the structure of oligosaccharides, various chromatographicand spectroscopic techniques were performed. High-performance anionexchange chromatography with pulsed amperometric detection (HPAEC-PAD)was employed. Using this technique, oligosaccharides were quicklyseparated into general groups based on charge (i.e., neutral, singlycharged, or multiply charged) and their structures were determined bycomparison to pure standards.

All methods were based on protocols described by Hardy and Townsend(Hardy, M. R., and Townsend, R. R., “High-pH anion-exchangechromatography of glycoprotein-derived carbohydrates”, 1994, MethodsEnzymol. 230: 208-225). Purified samples of transgenic avian derivedrhNaGlu were dialyzed using a Tube-O-Dialyzer against nanopure water at4° C. for about 24 hours to remove salts and other contaminants.Nanopure water was replaced four times during the entire dialysisperiod. After dialysis, each of the samples was divided into threealiquots. The aliquot intended for neutral and amino sugars analysis washydrolyzed with 2 N trifluoroacetic acid (TFA) at 100° C. for 4 hoursand the aliquot for mannose-6-phosphate analysis was hydrolyzed with6.75 N TFA at 100° C. for 1.5 hours. The hydrolysates were then driedunder N₂, re-dissolved with 50 μL H₂O, sonicated for 7 min in ice andtransferred to an injection vial.

A mix of standards for neutral and amino sugars, and formannose-6-phosphate with a known number of moles was hydrolyzed in thesame manner and at the same time as the sample. Four differentconcentrations of the neutral and amino sugar standard mix andmannose-6-phosphate were prepared to establish a calibration equation.The number of moles of each sugar in the sample was quantified by linearinterpolation from the calibration equation.

The oligossacharide profile and mannose-6-phosphate profile wereanalyzed separately by HPAEC-PAD. Instrument control and dataacquisition were accomplished using Dionex chromeleon software.HPAEC-PAD analysis of hydrolyzed rhNaGlu detected M6P. The mean measuredamount of M6P was 3.8 μg (CV 3.7%) per 210 μg of hydrolyzed protein.Converting to moles resulted in 13.4 nmol of M6P per 2.8 nmol of proteinwhich was equivalent to a ratio of 3.2 moles of M6P per mole of protein.

The oligosaccharide profile was also obtained for rhNaGlu (Gallus) usingHPAEC-PAD (see FIG. 8). The profiles demonstrated good repeatability ofthe PNGase F reaction on the single sample. Peak clusters were observedin regions corresponding to neutral oligosaccharides (˜10 min to ˜20min) A group of significantly smaller peaks eluting between ˜25 and ˜35min were also observed, which were possibly attributed to singly chargedspecies.

The monosaccharide composition analysis results obtained from samples ofrhNaGlu produced from a transgenic avian (Gallus) are summarized inTable 3, which tabulates the average molar ratio of each monosaccharideanalyzed for rhNaGlu.

TABLE 3 Monosaccharide Molar Ratios in rhNaGlu (Gallus)N-acetylgalactosamine (GalNAc)  1.1* N-acetylglucosamine (GlcNAc) 35.6*Galactose (Gal)   4* Mannose (Man) 25.5* Mannose-6-phosphate (M6P)  3.2*Fucose Not detected Glucose Not detected *mole of monosaccharide permole of protein

Example 4 Cellular Uptake into Fibroblasts

Wild-type human fibroblasts and mucopolysaccharidosis III B (NaGludeficient) human fibroblasts were placed in a 24-well plate (2.5×10⁴cells per well) and incubated for overnight at 37° C. in 5% CO₂.Conditioned media containing fibroblast basal medium and fibroblastgrowth kit having low serum were used. Various amounts of rhNaGlu (30,10, 3.0, 1.0, 0.3 and 0 μg/mL) were co-incubated for 24 hours at 37° C.with 5% CO₂ to determine levels of cellular uptake by the humanfibroblasts (see, FIG. 9). The wells were washed three times with PBS.100 μL lysis buffer was added per well and the plate was incubated for10 min at 37° C. Cell lysate was transferred into 1.5 mL centrifugetube. One cycle of freezing and thawing was performed. The cell lysatewas centrifuged at 10,000 rpm for 10 min 25 μL of supernatants were usedfor the assay. The assay time was 2 hours. The enzyme activity wasmeasured using the methods known in the art and according to the methodsdescribed in Marsh et al., Clinical Genetics (1985) 27: 258-262, Chow etal., Carbohydrate Research (1981) 96:87-93; Weber et al., ProteinExpression and Purification, (2001)21:251-259).

As shown in FIG. 9, negative control (i.e., MPS IIIB) did not exhibitany NaGlu activity while positive control (i.e., wild-type humanfibroblast) showed NaGlu activity. MPS IIIB cells treated with 0.3 μg/mLof rhNaGlu exhibited approximately 50% of the normal activity levelobserved in wild-type fibroblast cells. MPS IIIB cells treated with 1μg/mL of rhNaGlu demonstrated NaGlu activity that was approximately4-fold higher than that observed in wild-type cells. Surprisingly, MPSIIIB cells treated with 30 μg/mL of rhNaGlu showed NaGlu activity thatwas at least 40-fold higher than that observed in wild-type cells. Thisresult indicated that rhNaGlu produced from a transgenic avian (Gallus)was efficiently internalized into human fibroblasts at a high level.

To determine whether internalization of rhNaGlu is via M6P receptormediate endocytosis, M6P inhibition assays were performed. For the M6Pinhibition assays, various concentrations of free M6P were added tohuman MPS IIIB fibroblasts treated with 30 μg/mL of rhNaGlu andenzymatic activity was measured as described above. As shown in FIG. 10,human MPS IIIB fibroblasts did not exhibit any NaGlu activity,suggesting effective inhibition of NaGlu uptake by free M6P. Incontrast, MPSIII fibroblasts treated with 30 μg/mL of rhNaGlu in theabsence of free M6P exhibited a high level of enzymatic activity,suggesting that the protein was efficiently internalized into the NaGludeficient fibroblasts and retained activity. This enzymatic activity wasinhibited by the presence of M6P monosaccharide in the medium at theconcentration 0.03 mM and higher. The presence of 1 mM of M6Pmonosaccharide in conditioned medium inhibited more than 90% of cellularuptake of the protein.

These results indicated that the rhNaGlu produced from a transgenicavian was efficiently internalized into the MPS IIIB fibroblasts via M6Preceptor-mediated endocytosis and the rhNaGlu competed with M6Pmonosaccharides for the receptor recognition. The results wereconsistent with the glycan analysis that revealed the presence of theM6P structures on the rhNaGlu produced from the transgenic avian.

Example 5 Generation of Active NaGlu Fusion Proteins

Two different rhNaGlu fusion constructs were designed to validate thefeasibility of expressing rhNaGlu fusion proteins in the avianexpression system.

In one construct, a nucleic acid sequence encoding 8 consecutiveaspartic acid residues (DDDDDDDD) was fused to the nucleic sequenceencoding NaGlu protein at the 5′ end of the full-length NaGlu cDNAsequence (SEQ ID NO:2) using conventional PCR and DNA recombinanttechnology. In another construct, a nucleic acid sequence encoding TfRL(i.e., THRPPMWSPVWP; SEQ ID NO:5) was fused to the nucleic sequenceencoding NaGlu at the 3′ end of the full-length NaGlu cDNA sequence. Theeach construct was inserted into the pTT22 expression vector using EcoRIand HindIII restriction sites. The resulting vectors were eachtransfected into human embryonic kidney (HEK) 293 cells and stableclones expressing high levels of the fusion NaGlu proteins wereobtained. An rhNaGlu protein fused to a stretch of 8 consecutiveaspartic acid residues at N-terminus (AAA-NaGlu) and an rhNaGlu proteinfused to transferrin receptor ligand (TfRL) at C-terminus (NaGlu-TfRL)were isolated from conditioned media.

The enzymatic activity of AAA-NaGlu and NaGlu-TfRL was measured usingthe methods known in the art (see, e.g., Marsh et al., Clinical Genetics(1985) 27:258-262; Chow et al., Carbohydrate Research, (1981) 96:87-93;Weber et al., Protein Expression and Purification (2001) 21:251-259;Neufeld et al., Protein Expression and Purification (2000) 19:202-211;and Weber et al., Human Molecular Genetics (1996) 5:771-777.

As shown in FIGS. 13 and 14, AAA-NaGlu and NaGlu-TfRL fusion proteinsproduced from HEK293 cells showed high levels of enzymatic activity.These results confirmed the possibility that these constructs can beused to produce NaGlu fusion proteins that have increased levels ofphosphorylated mannose while retaining enzymatic activity from atransgenic avian expression system.

Example 6 Cellular Uptake into Macrophages

Internalization of rhNaGlu produced from Gallus into human macrophagecells was also measured. NR8383 macrophage cells were incubated with 10μg/mL of rhNaGlu in F12 growth media for 0, 4, 8, 24, 32 and 48 hours at37° C. with 5% CO₂. Samples were recovered and washed with PBS prior tolysis. 2.5×10⁵ cells were lysed in 1 mL of lysis buffer (10 mM of NaPhosphate pH6.0, 0.05% NP40), and lysates transferred into 1.5 mLcentrifuge tubes and centrifuged at 10,000 rpm for 10 min Proteinconcentration was determined by the Bradford assay and aliquots werefrozen for NaGlu enzyme assays.

Enzyme activity was measured using standard methods. 25 mM of substrate(4-methylumbelliferyl 2-Acetamido-2-deoxy-a-D-glucopyranoside) wasdiluted to 2 mM in nanopure water to form a working substrate stock.Dilutions of samples were prepared in assay buffer (1% bovine serumalbumin) 25 μL of 200 mM sodium acetate was distributed to wells of amulti-well plate. 25 μL of standard and 25 μL of samples were added todesignated wells. 50 μL of the working substrate stock was added to eachwell and the plate was gently tapped to mix. The plate was sealed withadhesive film and incubated at 37° C. for 30 minutes. The reaction wasthen terminated by addition of 50 μL of stop solution (1M Glycine pH12.5). The plate was placed on a microplate reader using a fluorescencebottom and the intensity was measured at an excitation 360 nm and anemission 460 nm. The level of liberated 4-methylumbelliferone (4-MU) wasmeasured by comparison with standards of 4-MU at 0.25 mM, 0.125 mM,0.0625 mM, 0.0312 mM, 0.0156 mM, and 0.0078 mM.

As depicted in FIG. 15, levels of the NaGlu activity in macrophagesincubated with 10 μg/mL of rhNaGlu increased almost linearly over a 48hour period: The rhNaGlu uptake by macrophages was rather slow, butsteady throughout the entire time period measured. The relatively slow,extended uptake of NaGlu activity (as compared to other lysosomalenzymes containing M6P and/or mannose in their glycosylation structures)was unexpected and surprising. Equally surprising and unexpected wasthat a large amount of rhNaGlu proteins was taken up into themacrophages over the extended time period, resulting in intracellularenzymatic activity levels at least 10, 50, 100, 200, 300, 500, or even1.000-fold higher than the basal levels observed in wild-typemacrophages not exposed to rhNaGlu. The results demonstrate that rhNaGluis extremely stable in extracellular as well as intracellularenvironments. Further, these results suggest that rhNaGlu may possessphysicochemical characteristics that allow for longer serum half-life(e.g., longer circulation) and high serum concentrations in vivo,properties which are ideal for enhanced uptake into the central nervoussystem (CNS).

TABLE 4 Summary of NaGlu Characteristics Avian (gallus) Natural producedhuman CHO produced rhNaGlu NaGlu human NaGlu Apparent Molecular ~85-~90~86  ~79-~89 Mass (kDa) Enzymatic Activity >1,000 ~500 ~1,057(nmol/min/mg) Mannose-6-phosphate High High None or very Low

Example 7 Administration of rhNaGlu into NaGlu Deficient Mice

Homozygous null mice were generated from breeding pairs of the strainB6.129S6-NaGlu^(tmlEfn)/J. Control wild-type mice were generated in thesame manner. Genotyping was performed according to a standard PCRprotocol. It is described in the art that at birth, homozygousnaglu(^(−/−)) null mice are viable, normal in size, and do not displayany gross physical or behavioral abnormalities, though they exhibited noNaGlu in all tissues (see, Li et al., (1999) Proc., Natl. Acad. Sci. USA96:14505-14510). At one month of age, vacuolated macrophages are foundin most tissues. Epithelial cells in kidney and neurons in some parts ofthe brain are also affected. The vacuolation becomes more prominent withage. At 4-5 months, the mice show abnormal behavior in an open fieldtest. Older animals may have urinary retention and difficulty walking.Typical life span of the homozygous null naglu^(−/−) mice is 8-12 months(see, Li et al., (1999) Proc., Natl. Acad. Sci. USA 96:14505-14510).

Intravenous (IV) Administration

The intravenous administration of test article and vehicle by tail veininjection was accomplished as follows. Before injection vasodilation wasachieved by gently warming the animal with an incandescent lamp or bysoaking the tail in warm water, approximately 43° C. The animal was thenplaced in restraint device. The surface of the tail was disinfected with70% isopropanol prior to injection. The lateral veins of the tail arelocated just under the skin and are identified in the distal part of thetail with the application of tension. A 27G needle, bevel up, wasinserted into the vein for 3-4 mm. The test article or vehicle was thenadministered as a slow bolus injection over a period of ten seconds asevidenced by the observed clearing of the vein as the administeredliquid momentarily occupies the vascular space. After removal of needle,gentle pressure was applied to the puncture site to provide hemostasis.The animal was monitored immediately following procedure to assurenormal activity.

Intrathecal (IT) Administration

The intrathecal administration of test article and vehicle by lumbarpuncture injection was accomplished as follows. Before injection,animals were anesthetized using isoflurane that was maintained via nosecone throughout the procedure. The site of injection was prepared byshaving the fur, as necessary, prior to each injection. The animal wasplaced in a prone position on a platform, ensuring the hind limbs werestraddling the platform forming a convex curve of the animals back. Thesurface of the back was swabbed with 70% isopropanol and allowed to dryprior to injection. Spinal column and hip bones were palpated to locatethe L4-L5 or L5-L6 margin. A 30G needle, bevel facing cranially, wasinserted into the intervertebral space. Placement was confirmed by theobservation of a tail flick. The test article or vehicle was thenadministered as bolus injection. The animal was allowed to recover fromanesthesia and monitored immediately following procedure to assurenormal activity and use of limbs.

Results

Twelve-week old naglu^(−/−) mice (B6.129S6-Naglu^(tmlEfn)/J) wereadministered rhNaGlu (Gallus) at dose levels of 6.75 or 27 mg/kg viatail vein injection (IV administration), once every other day, for atotal of 5 doses (at rhNaGlu concentrations of 1.125, or 4.5 mg/mL,respectively). Similarly, twelve-week old naglu^(−/−) mice wereadministered with rhNaGlu (Gallus) at a dose level of 0.31 mg/kg vialumbar puncture injection (IT administration), once every other day, fora total of 5 doses at NaGlu concentrations of 1.54 mg/mL. Vehicle (10 mMphosphate buffer, 150 mM NaCl and 0.02% Tween80, pH 5.5-5.8) wasadministered to naglu^(−/−) knock-out mice at the same doseconcentration for 5 doses every other day. Untreated wild-type andnaglu^(−/−) knock-out mice were also maintained for the duration of thestudy.

Animals were sacrificed 4 hours after the fifth and final injection. Allanimals were necropsied and the liver, brain, spleen, heart, lung andkidneys were excised. Each organ was divided sagittally, providingsamples for both frozen (−80° C.) and formalin-fixed storage.

Tissue samples were analyzed for: (1) heparan sulfate concentrationusing an analytical method based on SAX-HPLC analysis of heparan sulfatedisaccharides; and (2) α-N-acetylglucosaminidase enzyme activity using acell-based enzyme activity assay.

Histopathologic evaluation of brain, liver, kidney, spleen, heart andlung tissue was conducted using formalin-fixed tissue samples, embeddedin paraffin, sectioned at 4 μm, mounted on glass slides and stained withhematoxylin and eosin (H&E).

Following the repeated intravenous administration (5 doses over a 10 dayperiod) of rhNaglu (Gallus) to naglu^(−/−) mice at dose levels of 6.25and 27 mg/kg body weight, there was an apparent dose-dependent decreasein the concentration of Heparan Sulfate in the brain, liver and kidneyof naglu^(−/−) mice (Table 5; FIGS. 16-18). The relativea-N-acetylglucosaminidase activity was increased in the brain and liverfollowing intravenous administration (Table 6). These results wereunexpected and surprising because the NaGlu enzymatic activities andresulting substrate clearance were observed in the brain of the treatednaglu^(−/−) mice with the IV administration, suggesting that rhNaGlu(Gallus) administered systemically was distributed to the brain of thenaglu^(−/−) mice and effective to elicit efficacy even in the present ofthe blood brain barrier (BBB).

Following the intrathecal administration (5 doses over a 10 day period)of rhNaGlu (Gallus) to naglu^(−/−) mice at a dose level of 0.31 mg/kg,there was a decrease in the concentration of Heparan Sulfate in thebrain of naglu^(−/−) mice (Table 5; FIG. 19), suggesting that rhNaGlu(Gallus) was targeted to the brain and effective in reducing theaccumulated substrate in the brain of naglu^(−/−) mice.

TABLE 5 Tissue Substrate Level (rhNaGlu Gallus) Heparan Age at SulfateAnimal sacrifice Dose ug/mg Tissue Number Genotype (wks) Treatment(mg/kg) Route tissue mean sd KIDNEY 253 WT 4 na — — 0.1 155 WT 12 na — —0.045 0.0725 0.038891 178 KO 12 na — — 1.882 242 KO 4 na — — 1.687 145KO 13 na — — 1.904 474 KO 13 vehicle 0 IV 1.501 479 KO 13 vehicle 0 IV1.983 484 KO 13 vehicle 0 IV 1.839 1.799333 0.175908 487 KO 13 rhNaGlu6.25 IV 0.928 492 KO 13 rhNaGlu 6.25 IV 0.737 0.8325 0.135057 481 KO 13rhNaGlu 27 IV 0.591 485 KO 13 rhNaGlu 27 IV 0.311 490 KO 13 rhNaGlu 27IV 0.585 0.495667 0.159954 86 KO 15 vehicle 0 IT 2.105 91 KO 14 vehicle0 IT 1.704 1.9045 0.28355 94 KO 14 rhNaGlu 0.31 IT 1.324 101 KO 14rhNaGlu 0.31 IT 2.233 1.7785 0.64276 LIVER 253 WT 4 na — — 0.045 155 WT12 na — — 0.092 0.0685 0.033234 243 WT 4 na — — 0.045 178 KO 12 na — —1.85 242 KO 4 na — — 2.263 2.0565 0.292035 255 KO 4 na — — 1.85 474 KO13 vehicle 0 IV 1.822 479 KO 13 vehicle 0 IV 1.981 484 KO 13 vehicle 0IV 2.004 1.961667 0.165779 487 KO 13 rhNaGlu 6.25 IV 0.748 492 KO 13rhNaGlu 6.25 IV 0.444 504 KO 13 rhNaGlu 6.25 IV 0.494 0.562 0.163009 481KO 13 rhNaGlu 27 IV 0.491 485 KO 13 rhNaGlu 27 IV 0.172 0.3315 0.225567BRAIN 253 WT 4 na — — 0.021 155 WT 12 na — — 0.013 243 WT 4 na — —0.014308 10 WT 36 na — — 0.012649 0.015239 0.003906 239 KO 4 na — —0.095 178 KO 12 na — — 0.084 242 KO 4 na — — 0.099 255 KO 4 na — —0.094538 165 KO 24 na — — 0.084015 474 KO 13 vehicle 0 IV 0.085447 479KO 13 vehicle 0 IV 0.072 484 KO 13 vehicle 0 IV 0.073 0.085875 0.009972487 KO 13 rhNaGlu 6.25 IV 0.045 492 KO 13 rhNaGlu 6.25 IV 0.044119 504KO 13 rhNaGlu 6.25 IV 0.044 0.044373 0.000546 481 KO 13 rhNaGlu 27 IV0.017796 485 KO 13 rhNaGlu 27 IV 0.016668 490 KO 13 rhNaGlu 27 IV 0.0280.020821 0.006242 86 KO 15 vehicle 0 IT 0.094521 91 KO 14 vehicle 0 IT0.072623 0.083572 0.015484 94 KO 14 rhNaGlu 0.31 IT 0.038866 101 KO 14rhNaGlu 0.31 IT 0.028229 0.033548 0.007521 na: Not applicable (mice wereuntreated).

TABLE 6 Tissue enzymatic activity (rhNaGlu Gallus; U/ng protein)Enzymatic Age at Activity Animal sacrifice Dose (U/ug Tissue NumberGenotype (wks) Treatment (mg/kg) Route protein) BRAIN 253 WT 4 na — —7.7 178 KO 12 na — — 0 474 KO 13 vehicle 0 IV 0 479 KO 13 vehicle 0 IV 0484 KO 13 vehicle 0 IV 0.575 487 KO 13 rhNaGlu 6.25 IV 10.58 492 KO 13rhNaGlu 6.25 IV 5.066666667 504 KO 13 rhNaGlu 6.25 IV 4.033333333 481 KO13 rhNaglu 27 IV 87.91666667 485 KO 13 rhNaGlu 27 IV 90.15 490 KO 13rhNaGlu 27 IV 17.35 LIVER 253 WT 4 na — — 36.69 178 KO 12 na — — 0 474KO 13 vehicle 0 IV 0 479 KO 13 vehicle 0 IV 0 484 KO 13 vehicle 0 IV 0487 KO 13 rhNaGlu 6.25 IV 512.92 492 KO 13 rhNaGlu 6.25 IV 378.805 504KO 13 rhNaGlu 6.25 IV 607.9225 481 KO 13 rhNaGlu 27 IV 659.6825 485 KO13 rhNaGlu 27 IV 654.2475 490 KO 13 rhNaGlu 27 IV 677.8725 na: notapplicable (mice were untreated).

Each example in the above specification is provided by way ofexplanation of the invention, not limitation of the invention. In fact,it will be apparent to those skilled in the art that variousmodifications, combinations, additions, deletions and variations can bemade in the present invention without departing from the scope or spiritof the invention. For instance, features illustrated or described aspart of one embodiment can be used in another embodiment to yield astill further embodiment. It is intended that the present inventioncover such modifications, combinations, additions, deletions, andvariations.

All publications, patents, patent applications, internet sites, andaccession numbers/database sequences (including both polynucleotide andpolypeptide sequences) cited herein are hereby incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication, patent, patent application, internet site, oraccession number/database sequence were specifically and individuallyindicated to be so incorporated by reference.

1. A composition comprising an isolated mixture of recombinant humanN-acetyl-alpha-D-glucosaminidase (rhNaGlu) comprising the amino acidsequence 24-743 of SEQ ID NO:1, wherein at least 10% of said rhNaGlu insaid mixture comprises at least one glycan structure havingmannose-6-phosphate (M6P).
 2. The composition of claim 1, wherein saidrhNaGlu having M6P is capable of being taken up into a mammalian celldeficient in NaGlu such that internalized rhNaGlu restores at least 50%of normal NaGlu activity observed in a wild-type mammalian cell of thesame type.
 3. The composition of claim 2, wherein said glycan structureis an N-linked glycan.
 4. The composition of claim 3, wherein saidrhNaGlu contains at least 1 mole of M6P per mole of protein. 5.-10.(canceled)
 11. The composition of claim 2, wherein said mammalian celldeficient in NaGlu is a human cell.
 12. (canceled)
 13. The compositionof claim 11, wherein said human cell deficient in NaGlu is a neuronalcell.
 14. The composition of claim 13, wherein said rhNaGlu iseffectively delivered to the brain of a mammal having NaGlu deficiencywhen systemically administered. 15.-35. (canceled)
 36. The compositionof claim 1, wherein said rhNaGlu contains no fucose.
 37. The compositionof claim 1, wherein said rhNaGlu contains no glucose. 38.-43. (canceled)44. The composition of claim 1, herein said rhNaGlu is a fusion proteincomprising a second moiety. 45.-57. (canceled)
 58. A transgenic aviancomprising a transgene containing a promoter operably linked to anucleic acid sequence encoding a recombinant human NaGlu (rhNaGlu),wherein said transgene is contained in the genome of the transgenicavian and expressed in an oviduct cell such that said rhNaGlu isglycosylated in the oviduct cell of the transgenic avian, secreted intolumen of oviduct and deposited in egg white of an egg of the transgenicavian.
 59. The transgenic avian of claim 58, wherein said rhNaGlucomprises about 2, 3, 4 or 6 moles of M6P per mole of rhNaGlu.
 60. Thetransgenic avian claim 58, wherein said promoter component is anoviduct-specific promoter.
 61. The transgenic avian claim 60, whereinsaid oviduct-specific promoter is an ovalbumin promoter.
 62. (canceled)63. An egg produced by the transgenic avian of claim
 58. 64.-68.(canceled)
 69. A vector comprising a nucleotide sequence encoding ahuman NaGlu operably linked to an ovalbumin promoter.
 70. A host cellcomprising the vector of claim
 69. 71. (canceled)
 72. A pharmaceuticalformulation comprising a composition according to claim 1 in combinationwith a pharmaceutically acceptable carrier, diluent or excipient. 73.(canceled)
 74. A method of treating a subject suffering from NaGludeficiency, the method comprising administering to the subject atherapeutically effective amount of the composition of claim
 1. 75.-76.(canceled)
 77. The method of claim 74, wherein said NaGlu deficiency isSanfilippo B. 78.-88. (canceled)